HX00017361 

Biochemical  notes: 


LABORATORY  WORK 


[First  and  Second  PartsJ 


WILLIAM   J.   GIES 


XEW  YORK 

1906 


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BIOCHEMICAL   NOTES: 


LABORATORY   WORK 


[First  Part] 


BY 

WILLIAM   J.  GIES 


Col  an: 
College  of  PI:  ,.  .,-,,.     -,.,^ 

Libiraiy 


NEW  YORK 

1906 


Copyright,  1906 
By  WILLIAM  J.  GIES 


Press  or 
The  new  Era  Printins  Cohpanv 

lANCASTeil,  PA, 


PREFACE. 

This  volume  gives  a  bare  outline  of  the  laboratory  work  of  the 
course  in  physiological  chemistry  prescribed  for  first  year  students  of 
medicine,  at  Columbia  University,  during  the  second  half-year. 

The  work  of  preparing  the  volume  for  publication  was  begun  in 
January,  1906.  The  "first  part"  was  completed  before  the  be- 
ginning of  the  course  a  few  weeks  later.  It  is  my  intention  to 
prepare  and  issue  additional  parts  from  time  to  time,  as  more 
urgent  university  duties  will  permit. 

The  book  is  not  intended  to  reduce  in  any  degree  the  amount  of 
personal  instruction  heretofore  given  by  us,  to  each  member  of  the 
class,  without  the  help  of  such  printed  directions.  AVe  hope  it 
may  enable  us  to  increase  such  personal  attention.  The  student  is 
obliged  to  think  for  himself  as  much  as  possible,  under  guidance, 
and  is  required  to  prepare  a  complete  set  of  notes  on  the  phenomena 
brought  to  his  attention.  The  nature  of  the  directions  in  this  vol- 
ume and  our  discussions  of  the  experiments  before  and  after  com- 
pletion, are  expected  to  make  it  impossible  for  the  student  to  go 
through  his  work  mechanically.  The  laboratory  work  is  supple- 
mented by  lectures,  recitations  and  many  demonstrations. 

In  this  volume  the  capital  letter  P  is  used  to  signify  the  author's 
"Chemical  notes:  physical  and  inorganic'^  (1904).  The  numerals 
following  this  letter  are  intended  to  refer  to  sections  of  the  volume 
indicated.  The  capital  letter  0  is  used  in  the  same  way  to  desig- 
nate the  volume  of  "Chemical  notes:  or^a?n'c "  (1904-'05).  The 
capital  letter  L  refers  to  the  volume  of  "  Chemical  notes  :  labora- 
tory work''  (1905). 

William  J.  Gies. 
Laboratory  of  Physiological  Chemistry, 
College  of  Physicians  and  Surgeons, 
February  1,  1906. 


CONTENTS. 

[First  Paet.] 


Pagb 

Tables  showing  the  approximate  percentage  elementary 
composition  of  the  earth's  crust  including  air  and 
water,  and  of  a  man 5 

General  classification  of  the  substances  contained  in  plants 
and  animals,  with  the  names  of  representatives  op  each 

GROUP 6 

CHAPTER     I.     Inorganic  matters 7 

CHAPTER   II.     Fats .23 


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General  Classification  of  the  Substances  Contained 
Plants  and  Animals,  with  the  Names  of 
Representatives  of  Each  Group. 


IN 


Organic. 

Primary.     Synthetic  products  in  or- 
ganisms, made  in  plants  espe- 
cially : 
a.  Fats:      tripalmitin,     CjiHggOg ; 
fat-like     sxibstances ;    lecithin, 

C^HgoNPOg. 

h.  Carbohydrates :  stSiTch,  (CgH^QOs)! 
c.   Proteins:  albumin, 

^450^720-'^  116^6^1«' 

Secondary.  Analytic  products  in  the 
main,  which  are  wholly  or 
partly  derived  from  the  pri- 
mary by  metabolic  processes, 
in  animals  especially : 
a.  Aliphatic:  leucin, 
Nearly  all   the    inorganic    substances  re-  CrHinfNH.  )COOH. 

ferred  to  are  present  in  small  proportions  in        j.   Carbocyclie:  phenol,  CgHsOH. 
the  food  of  animals  and  many  are  formed  in       c.  Heterocyclic:  indol,  CgHjN. 
whole  or  part  in  animals  from  organic  sub-       d.  Products    of    unknown    chemical 
stances  by  analytic  processes.  character :  enzymes. 

Most  of  the  inorganic  substances  referred 
to  above  are  utilized  in  plants  in  processes 
resulting  in  the  synthesis  of  organic  sub- 
stances. 

This  simple  and  very  general  classification  should  be  kept  clearly 
in  mind  throughout  all  our  work  in  biological  chemistry. 


Inorganic. 
Water. 
Gases — ^free  and    in   solution :    carbon  di- 

oxid,  COj. 
Salts — precipitated  and  in  solution  : 

a.  Neutral :  sodium  chlorid,  NaCl. 

b.  Acid :     mono-potassium     di-hydrogen 

phosphate,  KHjPO^. 

c.  Alkaline  :  sodium  carbonate,  NajCO.,. 

d.  Cations  and  anions  of  all  the  inorganic 

substances  :  NH^  * ,  SO/''. 
Free  acids — in  solution  :  hydrochloric  acid, 

HCl ;  gaseous:  hydrogen  sulfid,HjS. 
Cations  and  anions  associated  with   organic 

radicals — Ca",  CjO/''. 


CHAPTER  I. 
INORGANIC  MATTERS. 

A.  Demonstrations.  Water  and  Inorganic  Solids  in 
Typical  Animal  and  Vegetable  Products,  such  as  Blood, 
Muscle  and  Potato  : 

I.  Distillation  of  contained  liquids  and  detection  of  water 
in  each  distillate.  2.  Quantitative  determination  of  water. 
3.  Inorganic  residue  obtained  by  incineration.  4.  Quantita- 
tive determination  of  ash.  5.  Distinction  between  preformed 
inorganic  matter  and  inorganic  matter  derived  from  organic 
matter  by  incineration. 

B.  Percentage  Amounts  of  Water  and  Ash  Obtained 
FROM  some  Mammalian  Parts. 

6.  In  the  subjoined  table  are  given  figures  representing  the  aver- 
age proportions  of  water  and  ash  obtainable  from  typical  mammalian 
liquids  and  solids  under  normal  conditions. 

Water.    Ash.  Water.  Ash 


Saliva 99.5  0.2 

Gastric  juice 97.3  0.7 

Bile 97.0  0.8 

Lymph 95.5  0.7 

Semen 90.3  0.9 

Kidney 81.1  1.0 

Blood  79.5  0.8 

Muscle 76.5  1.0 

Liver 76.2  1.0 


Spleen 72.0  0.6 

Brain  : 

Gray  matter 83.5  1.5 

White  matter 70.0  0.6 

Tendon 62.9  0.5 

Ligament 57.6  0.5 

Bone 30.0  43.0 

Dentin 10.0  66.0 

Adipose  tissue 4.4  0.2 


Cartilage 73.6      1.5    Enamel Trace    96.0 

C.  Leading  Inorganic  Ions  in  Biological  Liquids  : 

7.  Cations. — H',  NH/,  Na',  K*,  Ca",  Mg**. 

8.  Anions. — OH^  CV,  so/,  PO/^^  HPO/',  HjPO/,  COa^HCOj^ 


8  Biochemical  Notes. 

D.  Methods  foe  the  Detection  of  the  Most  Important 
Inorganic  Cations  and  Anions  in  Typical  Biological 
Liquids  *  such  as  Blood,  Urine  and  Fruit  Juice,  f     (L  :  92 

-i92.)1: 

9.  Hydrion,  H*,  and  hydroxidion,  OH'.  Determine  the  reac- 
tions (L :  5S-65)  of  the  biological  liquids  to  litmus,  lacmoid  and 
phenolphthalein  (67). 

10.  Metals  (cations).  With  the  exception  of  iron,  heavy 
metals  occur  only  very  rarely,  or  in  only  very  minute  proportions 
in  biological  products,  and  special  methods  as  well  as  relatively  large 
quantities  of  material  are  required  for  their  detection.  Wherever 
in  normal  biological  products  iron  and  other  heavy  metals  may  be 
found,  the  metallic  atoms  exist,  as  a  rule,  in  complex  molecular 
combinations  and  occur  very  rarely  in  ionic  or  dissociable  forms.  § 

*  Biological  solids  may  ordinarily  be  dissolved  without  particular  difficulty. 
Aqueous  or  acid  solutions  or  extracts  usually  contain  ions  fully  representative  of 
all  the  inorganic  substances  present  in  the  original  material.  See  the  succeeding 
footnote. 

t  The  plan  of  qualitative  analysis  presented  in  this  chapter  -will  enable  the  stu- 
dent to  prove  definitely  the  absence  of  some  common  cations  and  anions,  as  vcell 
as  to  demonstrate  the  presence  of  certain  others.  This  intention  has  prevented  the 
brevity  that  might  otherwise  have  been  sought  in  the  scheme  of  processes  here 
outlined.     See  32-35, 

X  See  preface, 

§  Various  organic  substances  of  common  occurrence  in  biological  products,  such 
as  oxalic  acid,  citric  acid,  tartaric  acid,  sugars,  purin  bases,  and  proteins,  inter- 
fere more  or  less  with  the  detection  of  certain  cations,  especially  of  some  of  the 
heavy  metals.  Thus,  hydrochloric  acid  precipitates  a  number  of  proteins,  e.  g., 
mucoids,  in  masses  that  resemble  superficially  the  precipitates  of  the  metals  of  the 
first  group.  Oxalic  acid  interferes  with  the  precipitation  of  tin  as  sulfid  by  hydro- 
gen sulfid  and  favors  the  precipitation  of  earthy  metals  as  oxalates,  with  metals 
of  the  third  group  as  hydroxids,  when  their  mixed  solutions  are  rendered  alkaline 
with  ammonium  hydroxid  (15),     Such  instances  might  be  multiplied. 

When  it  is  desired  to  detect  traces  of  heavy  metals  as  cations,  or  larger  propor- 
tions "combined,"  in  biological  materials,  it  is  necessary  as  a  preliminary  step 
in  the  application  of  the  customary  methods  of  inorganic  analysis  to  destroy  the 
organic  matter.  This  may  be  accomplished  in  several  ways.  Thus,  the  product 
may  be  acidified  strongly  with  nitric  acid,  the  acidified  material  evaporated  to 
dryness  and  the  dry  residue  ignited  very  gently  until  no  further  odor  of  burning 
material  can  be  detected.  The  nitric  acid  favors  the  conversion  of  any  iron  to  the 
ferric  form  and  aids  in  the  destruction  of  the  organic  matter.  Complete  char- 
ring is  sufficient  at  this  stage  and  high  heat  must  be  avoided  in  order  to  prevent 
conversion  of  iron,  particularly,  into  a  very  insoluble  oxid.  The  black  residue 
should  be  thoroughly  extracted  with  warm  nitric  acid  and  the  extract  filtered. 
The  washings  should  be  added  to  the  main  filtrate.     The  carbonaceous  matter  on 


Inorganic  Matters.  9 

1 1 .  First  group.  To  about  5  c.c.  of  the  original  (filtered  ?)  liquid 
add  drop  by  drop  a  moderate  excess  of  dilute  hydrochloric  acid. 
Preserve  the  liquid  for  use  in  process  I2.  Hydrochloric  acid  and 
other  acids  precipitate  a  number  of  common  organic  biological  com- 
pounds,* to  which  attention  will  be  drawn  later.  See  footnote, 
page  8.     Silicates  are  rarely  if  ever  present  in  biological  liquids 

the  filter  may  be  completely  incinerated  at  a  relatively  low  temperature,  with 
the  aid  of  nitric  acid,  and  a  second  warm  acid  extract  obtained  for  the  recovery 
of  residual  traces,  which  should  be  added  to  the  previous  extract.  A  silicious 
residue  may  remain.  The  combined  acid  extracts  should  be  evaporated  to  com- 
plete dryness  on  a  water-lath,  until  no  further  odor  of  nitric  acid  can  be  detected 
on  stirring.  Ignition  must  be  avoided.  The  residue  should  be  treated  with  a 
little  concentrated  nitric  acid,  diluted  with  water  and  boiled.  The  residue  dis- 
solves completely,  or  a  white  deposit  of  silicic  oxid  remains.  To  the  combined 
filtrate  and  washings  ammonium  hydroxid  should  be  added  until  the  solution 
is  rendered  neutral  or  very  nearly  neutral.  To  this  solution  may  be  applied 
directly  the  successive  operations  outlined  above  (11-35),  except  those  for  the 
detection  and  removal  of  oxalic  acid.  More  detailed  methods  than  those  given 
above  (11-35)  must  be  used  for  the  identification  of  metals  of  unusual  biological 
occurrence  that  might  be  present.  Such  methods  are  given  in  the  author's  volume 
on  laboratory  work  in  general  chemistry  (L ;  92-158),  and  in  most  volumes  on 
qualitative  inorganic  analysis. 

The  method  of  eliminating  organic  matter  that  has  just  been  described  usually 
results  in  the  production  of  inorganic  matter  from  organic  non-electrolytes,  and 
on  that  account  does  not  give  a  true  indication  of  the  character  of  preexistent 
ions  or  dissociable  compounds  when  they  occur.     (Demonstration  5. ) 

*  If  a  precipitate  was  produced  by  hydrochloric  acid,  filter  and  subject  the  pre- 
cipitate to  the  following  treatment  in  order  to  show  the  presence  or  absence 
of  metals  of  the  first  group  —  silver,  mercury  (ous),  lead.  Wash  slightly  with 
cold  water.  Reject  the  washings.  Treat  the  precipitate  on  the  filter  with  about 
25  c.c.  of  hot  water.  Pour  the  filtrate  on  the  precipitate  several  times  (boil  each 
time  before  doing  so)  to  insure  complete  solution  of  any  lead  chlorid  that  may  be 
present.  Filter.  Cool.  Test  for  lead  in  the  filtrate  by  adding  potassium  iodid 
or  dilute  sulfuric  acid.     (L:  105.) 

Wash  the  residue  with  hot  water.  Eeject  the  washings.  Treat  the  residue 
on  the  filter  with  about  25  c.c.  of  warm  ammonium  hydroxid.  Pour  the  filtrate 
upon  the  precipitate  several  times  (warm  each  time  before  doing  so)  to  insure 
complete  removal  of  any  silver  chlorid  that  may  be  present.  Merourous  chlorid 
is  converted  by  this  treatment  into  a  black  solid  mixture  of  metallic  mercury  and 
mercuric  ammonium  chlorid.  A  white  residue  at  this  point  probably  consists  of 
albuminous  matter.  Filter.  Any  silver  chlorid  originally  present  in  the  pre- 
cipitate would  be  contained  in  the  ammoniacal  filtrate  in  the  form  of  ammonio- 
silver  chlorid.  Albuminous  matter  would  also  dissolve  somewhat.  Acidify  the 
filtrate  with  nitric  acid.  If  a  white  precipitate  is  formed  in  the  acid  liquid,  silver 
as  silver  chlorid  is  indicated.  Silver  chlorid  gradually  darkens  in  diffused  sun- 
light and  does  not  blacken  on  ignition.  A  white  precipitate  of  albuminous 
matter  that  might  occur  at  this  point  is  not  affected  by  sunlight  but  blackens  and 
disappears  on  ignition.     (L  :  105.) 


10  Biochemical  Notes. 

in  sufficient  proportions  for  silicic  acid  to  be  precipitated  by  hydro- 
chloric acid.     See  39. 

If  a  precipitate  was  produced  on  adding  hydrochloric  acid,  observe 
whether  sodium  chlorid  or  any  other  soluble  chlorid  brings  about 
the  same  result  in  a  new  portion  of  the  liquid.* 

12.  Second  group.  As  a  rule  nearly  all  the  metals  of  the 
second  group  are  entirely  absent  from  normal  biological  liquids. 
Although  traces  of  arsenic,  for  example,  appear  to  be  normally 
present  in  practically  all  animals  and  copper  is  contained  in  many, 
the  proportions  in  which  these  elements  occur  are  too  trifling  for 
detection  by  ordinary  means  (footnote,  page  8).  Besides,  wherever 
arsenic  and  copper  exist  in  animals  ordinarily,  they  occur  as  a  rule  in 
organic,  non-dissoeiable  combinations. 

13.  Warm  the  clear  acid  liquid  (ll),  or  the  filtrate  from  (or  ex- 
tract of)  any  precipitate  that  may  have  been  formed  by  hydrochloric 
acid.  Add  a  drop  of  hydrochloric  acid  to  make  certain  that  pre- 
vious (?)  precipitation  was  complete,  filter  if  necessary  and  treat  the 
solution  with  a  slight  excess  of  hydrogen  sulfid,  preferably  in  aqueous 
solution.     Cool.     Proceed  to  14. 

14.  Third  group.  Preliminary  examination  for  phosphate  and 
oxalate.  Phosphate  occurs  almost  universally,  oxalate  occasionally, 
in  biological  materials.  Oxalate  is  always  accompanied  in  organ- 
isms by  phosphate.  The  presence  or  absence  of  phosphates  and 
oxalates  determines,  as  a  rule,  the  method  to  be  employed  for  the 
detection  of  the  metals  of  the  third,  fourth  and  fifth  groups.f 

15.  PreGipitation  of  phosphates  and  oxalates.  To  about  25  c.c. 
of  the  original  solution  %  add  a  few  drops  of  dilute  nitric  acid  and  boil 
for  about  a  minute.     Filter  off,  wash   and  reject  any  precipitate 

*  Any  albuminous  matter  that  might  be  precipitated  by  hydrochloric  acid  would 
remain  in  solution  on  addition  of  sodium  chlorid  instead  of  hydrochloric  acid.  On 
the  contrary  the  metals  of  the  first  group  would  be  precipitated.  See  footnote,  page  9. 

t  Silicates  and  fluorids,  which  occasionally  occur  in  minute  proportions  in 
biological  liquids,  affect  slightly  the  method  of  procedure  at  this  point.  Their 
influence  need  not  be  taken  into  account,  however,  except  in  very  special  cases. 

%  Blood  and  other  albuminous  liquids  should  be  diluted  with  several  volumes 
of  water  before  adding  the  nitric  acid.  If  any  of  the  metals  of  the  first  or  second 
groups  were  detected  in  tests  11  and  13  they  must  be  removed  from  the  solution 
before  proceeding  to  the  separation  of  the  metals  of  the  third,  fourth  and  fifth 
groups.  After  the  elimination  of  such  heavy  metals  hydrogen  sulfid  should  be 
compUlely  removed  from  the  final  filtrate  by  boiling.  Determine  this  matter 
accurately  by  testing  the  vapor  with  wet  "lead  acetate  paper."  The  solution 
is  then  in  proper  condition  for  the  treatment  indicated  above,  under  15. 


Inorganic  Matters.  11 

(albuminous)  that  may  be  produced  at  this  point.  Treat  the  acid 
solution  (filtrate?)  with  about  an  equal  volume  of  ammonium  chlorid 
solution  and  a  slight  excess  of  ammonium  hydroxid.  Boil  until  the 
odor  of  ammonia  can  hardly  be  detected.  Filter  the  slightly  alkaline, 
precipitated  mixture.*  Wash.  Retain  the  filtrate  and  washings  Jor 
examination  later  (24)  and  proceed  with  the  precipitate  as  follows 
(16-19). 

16.  Detection  of  phosphate.  Dissolve  a  small  portion  of  the  pre- 
cipitate in  a  little  nitric  acid.  Add  about  an  equal  volume  of 
"molybdic  solution"  (ammonium  molybdate  in  nitric  acidf). 
Warm  gently  to  about  the  temperature  of  the  body  and  let  the 
mixture  stand  under  observation  until  after  the  conclusion  of  the 
next  test.     Typical  equation  : 

12NH4HMo04  +  NajHPO^  +  IIHNO3  =  (NHj3PO«(Mo03),j  +  9NH4NO3  +  2NaN03  +  12H,0 

Ammonium  phospho- 
molybdate  (yellow) 

If  an  appreciable  quantity  of  phosphate  is  present,  a  yellow  pre- 
cipitate will  form  in  a  few  minutes. | 

17.  Detection  of  oxalate.  Treat  a  portion  of  the  precipitate  with 
a  moderate  excess  of  sodium  carbonate  and  boil  for  a  minute  or  two. 
Filter.  Acidify  the  filtrate  slightly  with  acetic  acid  and  add  cal- 
cium chlorid.     Typical  equation  : 

CaClj  +  Na^CjOi  =  CaQO,  +  2NaCl 
Calcium 
oxalate 
(white) 

The  white  pulverulent  precipitate  of  calcium  oxalate  forms 
quickly  in  the  presence  of  even  traces  of  oxalate. 

*The  precipitate  obtained  at  this  point  in  biological  liquids  usually  consists 
chiefly  of  earthy  phosphates,  almost  invariably  of  calcium  phosphate  and  am- 
monio-magnesium  phosphate  ;  but  inorganic  iron,  as  well  as  manganese  and  other 
metals  of  the  third  and  fourth  groups  that  might  have  been  contained  in  the  original 
solution,  would  also  be  present  as  hydroxid  or  phosphate  or  both  ;  and,  if  oxalate 
occurred  in  the  original  solution,  earthy  oxalates,  chiefly  calcium  oxalate,  might 
also  be  contained  in  the  precipitate.  See  the  footnote  on  page  8.  Silicates 
are  present  in  biological  liquids  in  proportions  that  are  too  slight  to  yield  precipi- 
tates of  silicic  acid  on  adding  ammonium  hydroxid  (39). 

t  Ammonium  molybdate  solution  or  so-called  "molybdic  solution''  is  usually 
made  in  the  following  proportions  :  100  grams  of  molybdic  anhydrid  are  dissolved 
in  417  c.c.  of  ammonium  hydroxid  (sp.  gr.  0.96).  This  solution  is  poured  in  small 
quantities  at  a  time  into  1250  c.c  of  nitric  acid  (sp.  gr.  1.20).  The  acid  solution 
is  thoroiighly  shaken  after  each  addition  of  the  alkaline  liquid.  The  mixture  is 
kept  in  a  warm  place  for  several  days.     The  filtrate  is  the  reagent. 

X  Arsenate  could  not  be  present  in  the  solution.     See  13,  also  45. 


12  Biochemical  Notes. 

18.  Detection  of  metals  of  the  third  group  (iron).  Of  these  iron 
is  the  only  one  of  particular  biological  importance.  It  can  seldom 
be  detected  directly  in  biological  liquids. 

19.  If  oxalate  was  absent  (17)  proceed  to  20.  If  both  oxalate 
and  phosphate  were  detected  (16-17)  in  the  precipitate  (15)?  the 
remaining  portion  of  the  precipitate  (with  the  filter  paper  if  neces- 
sary) should  be  transferred  to  a  porcelain  crucible  or  a  platinum 
foil,  dried  and  very  gently  ignited.  The  oxalic  acid  will  be  de- 
stroyed by  combustion.     Proceed  to  20. 

20.  This  ignited  residue  (19),  or  the  main  bulk  of  the  original 
precipitate  (15)  if  oxalate  was  absent,  should  be  dissolved  in  a 
small  amount  of  hydrochloric  acid.     Proceed  to  21. 

21.  Iron.  Dilute  with  water  a  small  quantity  of  the  acid  solu- 
tion just  prepared  (20)  and  add  to  it  potassium  ferrocyanid  or  am- 
monium sulfocyanate.  As  a  rule  the  test  is  negative,  unless  an  ash 
is  the  material  undergoing  analysis.  Use  the  main  bulk  of  the 
solution  in  process  22.     Equations  : 

a.  4FeCl3  +  3K,Fe(CN)6  =  Fe4[Fe(CN)fil3  +  12KC1 

Ferric  ferrocyanid 
("  Prussian  blue") 

b.  FeCls  +  3NH4SCN  =  Fe(SCN)3  +  3NH4CI 

Ferric  sulfo- 
cyanate 
(red:  soluble) 

22.  Removal  of  metals  of  the  third  group  (iron),  and  also  of 
phosphate.  Add  sodium  hydroxid  to  the  remainder  of  the  acid 
solution  (20)  until  the  latter  is  nearly  neutral.  Treat  the  resultant 
slightly  acid  liquid  with  a  solution  of  sodium  acetate  strongly 
acidified  with  acetic  acid  and  heat  the  mixture  gently  for  about  10 
minutes,  to  remove  excess  of  acetic  acid.  If  iron  was  present  fer- 
ric phosphate  will  be  precipitated.  To  the  solution  add  ferric 
chlorid  drop  by  drop  until  the  liquid  assumes  a  red  color.  This 
coloration  indicates  that  the  iron  which  was  introduced  has  united 
with  all  the  phosphate  in  the  solution  and  has  begun  to  combine 
with  the  associated  acetate.  Heat  gently  for  about  10  minutes. 
Filter  while  the  mixture  is  warm.  Wash  with  hot  water.  Reject  the 
precipitate,  which  consists  of  phosphate  and  acetate  of  iron.*  The 
filtrate  and  washings  may  contain  cations  of  any  metals  of  groups 

*Much,  if  not  all,  the  iron  obtained  at  this  point,  it  will  be  recalled,  was 
purposely  added  to  the  solution  for  the  removal  of  phosphate. 


Inorganic  Matters.  13 

four  and  five  that  were  contained  in  the  original  precipitate  (15). 
It  may  also  contain  a  trace  of  iron  that  was  not  precipitated  in  the 
foregoing  process.  A  method  for  removing  such  a  trace  of  iron  is 
indicated  below  (24).     Proceed  to  24. 

23.  Fourth  group.  Of  this  group  manganese  is  the  only  one 
of  general  biological  significance.  It  can  seldom  be  detected  by 
ordinary  means  because  of  the  slight  proportions  in  which  it  oc- 
curs. Like  iron  it  occurs  mainly  if  not  wholly  in  organic  combi- 
nation.    (See  footnote,  page  8.) 

24.  Mix  filtrates  15  and  22.  Reduce  the  volume  one  half  by 
evaporation.  Add  to  the  concentrated  liquid  a  moderate  excess  of 
ammonium  hydroxid,  and  boil  until  nearly  all  ammonia  has  been 
eliminated.  Filter,  if  any  trace  of  iron,  or  anything  else  that 
had  not  been  previously  precipitated,  should  be  thrown  out  of 
solution  at  this  point.  Add  colorless  ammonium  sulfid,  in  slight 
excess.     Heat  to  boiling.     Filter  if  necessary.*     Proceed  to  26. 

25.  Fifth  group.  Of  this  group  calcium  is  the  only  element 
of  biological  importance.  It  is  a  relatively  prominent  constituent 
of  practically  all  organisms. 

26.  Calcium.  The  preceding  filtrate  (24)  should  be  concen- 
trated by  evaporation  and  cooled.  Excess  of  ammonium  sulfid  is 
expelled  in  this  process.  If  a  precipitate  forms  as  a  result  of  this 
treatment  add  just  enough  water  to  dissolve  the  precipitate. f 
Render  the   liquid  alkaline  with  ammonium   hydroxid  and  add 

*  As  a  rule  precipitation  does  not  occur  because  of  the  absence  of  manganese 

and  the  remaining  metals  of  the  fourth  group  or  because  only  the  merest  traces 

are  present.     If  a  precipitate  is  produced,  however,  test  for  manganese  as  follows  : 

Dissolve  the  precipitate  on  the  filter  in  dilute  hydrochloric  acid.     To  the  filtrate 

add  excess  of  sodium  hydroxid.     If  manganese  was  present  it  will  be  precipitated 

as  white  manganous  hydroxid  which  will  gradually  be  converted  into  the  brown 

manganic  hydroxid.     Put  the  filter  and  precipitate  in  a  casserole  and  heat  to 

boiling  in  nitric  acid.     Add  several  very  small  quantities  of  red  lead  (less  than 

half  a  gram  in  all).     Boil  again  for  a  moment.     Filter.     A  pink  filtrate  indicates 

the  presence  of  manganese.     This  color  must  be  looked  for  immediately  after 

filtering.     When  only  a  small  proportion  of  manganese  is  present  the  faint  pink 

color  speedily  disappears  as  a  result  of  oxidation.     The  color  may  usually  be  seen 

on  permitting  the  excess  of  PbjO^  to  subside.     (L  :  138.)     Equation  : 

2Mn(0H)j  +  bVhf)^  +  SOHNO,  =  2HMnO,  4-  15Pb(NOs)2  +  16H.0 

Permanganic 
acid  (red) 

If  manganese  is  present  in  the  precipitate  produced  by  ammonium  sulfid,  the 
precipitate  will  impart  an  amethyst-red  color  to  a  borax  bead  when  it  is  heated 
with  the  latter  on  a  platinum  wire  in  an  oxidizing  flame.  In  a  reducing  flame  the 
bead  becomes  colorless. 

t  If  sulfur  s^'parates  filter  the  mixture. 


14  I  Biochemical,  Notes. 

ammonium  carbonate  to  it  until  precipitation  is  complete.  Warm 
the  mixture  but  do  not  heat  to  boiling.  Filter  off  the  precipitate 
of  calcium  carbonate  and  use  the  filtrate  for  the  detection  of  mag- 
nesium in  process  28.  Confirm  the  presence  of  calcium  in  the 
washed  precipitated  carbonate  by  dissolving  the  precipitate  in  a 
slight  quantity  of  acetic  acid.  In  this  acid  liquid,  ammonium  oxa- 
late causes  precipitation  of  calcium  oxalate  (17)'  Let  the  mixture 
stand  if  precipitation  does  not  occur  immediately.     Equation  (17) : 

CaCCHjCOj)^  +  (NHJ2C2O4  =  CaCp,  +  2NH,CH3C02 

Calcium 
oxalate 
(white) 

27.  Sixth  group.  Magnesium,  sodium,  potassium  (and  am- 
monium) are  present  in  practically  all  biological  products. 

28.  Magnesium,  Concentrate  by  evaporation  the  filtrate  from 
the  precipitated  carbonate  (26),  and  add  moderate  excesses  of  am- 
monium chlorid  and  ammonium  hydroxid.  Treat  the  solution  with 
ammonium  oxalate.  Let  the  mixture  stand  several  minutes.  Filter 
off  any  calcium  oxalate  that  may  have  been  precipitated.  *  Add 
drop  by  drop  to  the  filtrate  a  moderate  excess  of  di-sodium  hydrogen 
phosphate.  Stir  thoroughly.  Let  the  mixture  stand  a  few  minutes. 
Equation  : 

MgCl^  +  Na^HPO,  +  NH,OH  =  NH.MgPO,  +  2NaCl  +  H,0 

Ammonio-mag- 

nesium  phosphate 

(white) 

In  each  of  the  remaining  tests  for  cations  (29-30)  use  portions 
of  the  original  solution,  as  follows  : 

29.  Sodium  and  potassium.  Flame  tests.-\  Use  platinum  wire. 
Persistent  yellow  indicates  sodium.  A  violet  color,  not  obscured 
by  blue  glass,  indicates  potassium. 

30.  Ammonium.  Add  sodium  hydroxid  in  excess.  Boil.  No- 
tice the  character  of  any  (a)  odor  that  may  be  detected,  also  the 
reaction  of  the  steam  on  (6)  wet  litmus  paper,  and  the  effect  of  the 
steam  on  (c)  hydroohloric  acid  adherent  to  a  glass  rod.     Equations  : 

a.  NH.Cl     +    NaOH    =    NH3     +     H^O    +    NaCl 

Ammonia 

(gas) 

c.  NH3        +        HCl        =        NH.Cl 

Ammonium  chlorid 
(white  fumes) 

*  Calcium  is  not  completely  precipitated  by  ammonium  carbonate, 
t  Special  methods  are  required  in  unusual  cases.     Sodium  and  potassium  are 
invariably  present  in  biological  products. 


Inorganic  Matters. 


15 


31.  Summary  of  cation  processes,  9-30.  In  the  subjoined 
summary  the  methods  heretofore  described  for  the  detection  of 
cations  are  indicated  in  a  manner  intended  to  favor  easy  review. 


I.  Original  Solution  +  HCl,  etc.  (11).     Filter  (?)  : 


Pbecipitate  (?)     Filtrate  4- H2^)  etc.  (13).     Filter  (?)  : 
See  II.  1 


II.  Original  Solution. 
Filter  : 


Precipitate  (?)     Filtrate 
See  13.  Eeject. 

Process  15  (precipitation  of  phosphates  and  oxalates). 

I 


Precipitate  (15) 

A.  Preliminary  tests: 

((..  Phosphate,  test  16. 
h.  Oxalate,  test  17. 

B.  Solution  of  precipitate  (19-20) 
Test  for  iron,  (?)  21 


Filtrate  (i5)  +  (NHi)2S,  etc.  (24) 
Filter  : 


Precipi- 

-  ,.  K    .  I        TATE  (?) 

b.  Removal  of  iron  and  phos«]j     Manga- 


F1LTRATE+  (NHJjCOj, 
etc.  (26)  Filter: 


phate,  22.     Filter: 


Precipitate  Filtrate  (22)  s- 
Keject.  Combine  with 

filtrate  15. 


nese (?) 
Test  24. 


Precipitate    Filtrate +  Na2HP04, 
Calcium.  etc.  (28).    Filter: 

Test  26.  I 


Precipitate 
Magnesium. 
Test  28. 


Filtrate 
Eeject. 


III.  Original  Solution. 

A.  Flame  tests,  29  :  Sodium,  potassium. 

B.  Ammonia,  30  :   Ammonium. 


G 
O 

<J 

bo 


s  s 


o 
U 


Methods  for  the  detection  of  cations  of  metals  of  groups  V 
and  VI  in  the  absence  of  cations  of  metals  of  groups  I-IV.  32. 
Since  calcium,  magnesium,  sodium,  potassium  and  ammonium  are 
the  leading  inorganic  cations  contained  in  biological  liquids,  and 
since  the  cations  of  the  metals  of  groups  I-IV  are  usually  absent,  the 
following  plan  of  analysis  leads  to  a  more  speedy  detection  of  the 
cations  of  greatest  biological  importance. 

33.  Test  for  sodium,  ijoiassium  and  ammonium  as  was  previously 
indicated  (29-30). 


16  Biochemical  Notes. 

Test  for  calcium  and  magnesium  as  follows :  34.  Calcium. 
Make  the  solution  alkaline  with  ammonium  hydroxid.  Earthy 
phosphate  may  be  precipitated.  To  this  mixture  add  acetic  acid 
drop  by  drop  until  the  reaction  of  the  liquid  is  acid  and  all  the 
precipitated  phosphate  has  gone  into  solution.  Filter  if  necessary. 
Treat  the  acid  mixture  with  ammonium  oxalate.  Calcium  oxalate 
will  be  precipitated  (17).  Heat  the  mixture  to  boiling  in  order  to 
favor  filtration.  Filter.  (Test  the  filtrate  with  ammonium  oxalate 
to  make  certain  that  calcium  was  completely  precipitated  in  the  pre- 
ceding treatment.) 

35.  Magnesium.  To  the  filtrate  (34)  add  excess  of  ammonium 
hydroxid  and  di-sodium  hydrogen  phosphate.*  A  white  precipi- 
tate of  ammonio-magnesium  phosphate  is  usually  formed  (28). 

36.  Acids  (anions).  The  salts  that  are  of  particular  biological 
significance  and  which  are  represented  by  the  corresponding  in- 
organic anions  are  chlorids,  phosphates,  carbonates  and  sulfates. 
Silicates,  fluorids  and  sulfids  are  less  conspicuous.  Trifling  quanti- 
ties of  sulfocyanates,  nitrates,  nitrites  and  other  inorganic  salts  also 
occur  normally  in  various  biological  products. 

37.  Boil  about  25  c.c.  of  the  original  solution  f  with  a  moderate 
excess  of  sodium  carbonate.  |  If  a  precipitate  is  formed  at  this 
point  it  may  contain  carbonates,  hydroxids,  phosphates,  silicates 
and  fluorids.  Filter,  if  necessary,  while  the  mixture  is  hot,  and  in 
that  case  use  the  filtrate  as  indicated  below  (41-47)  and  the  pre- 
cipitate as  follows  : 

38.  Transfer  all  the  precipitate  (37)  §  to  an  evaporation  dish, 
add  sufficient  nitric  acid  to  acidify  the  resultant  solution  and 
evaporate  it  to  dryness  on  a  water  bath.  Treat  the  residue  (39)  on  a 
hot  water  bath  with  dilute  nitric  acid  and  water,  filter  and  wash  (40). 

39.  Silicate.  A  white  ^n%  residue  at  this  point  (38)  consists  of 
hydrated  silicic  acid.     See  43.     A  sandy  residue  rarely  remains  at 

*  There  may  be  sufficient  P04iQ  the  filtrate  to  make  this  addition  of  phosphate 
unnecessary  and  to  yield  a  precipitate  of  ammonio-magnesium  phosphate  on  ad- 
dition of  the  ammonium  hydroxid. 

f  See  footnote,  page  11. 

X  Heavy  metals  tend  to  prevent  easy  detection  of  some  anions.  Any  heavy 
metals  present  in  the  liquids  under  examination  are  precipitated  by  the  carbonate. 
As  a  rule  this  process  is  unnecessary  because  of  the  absence  of  cations  of  heavy 
metals.     (11-13.) 

2  A  trace  of  fluorid  may  be  present  in  the  precipitate.  When  its  detection  is 
sought,  a  portion  of  the  precipitate  must  be  reserved  for  special  tests  for  fluorin. 


Inorganic  Matters.  17 

this  stage  of  the  process.     Any  albuminous  matter  that  might  be 
present  would  disappear  on  ignition  ;  the  silicic  acid  would  remain 

(43). 

40.  Phosphate.     Test  the  filtrate  (38)  for  phosphate  as  in   16, 

but  filter  before  adding  molybdic  solution,  if  the  nitric  acid  produced 
a  precipitate. 

41.  Divide  into  three  unequal  parts  the  unprecipitated  alkaline 
solution  obtained  after  the  above-described  treatment  with  sodium 
carbonate  (37),  or  the  alkaline  filtrate  from  any  precipitate  that 
may  have  been  formed.  Tests  should  be  applied  to  the  parts  as 
follows  : 

First  part  (|).  Add  a  few  drops  of  nitric  acid.  Boil  to  expel 
carbon  dioxid.  Add  more  nitric  acid,  if  necessary,  to  effect  per- 
manent slight  acidification.  Albuminous  matter  may  be  precipitated. 
Filter  if  necessary  and  reject  the  precipitate.  Then  add  ammonium 
hydroxid  to  make  the  reaction  slightly  alkaline.  Boil  until  the 
odor  of  ammonia  can  no  longer  be  detected  in  the  vapor.  Filter  if 
necessary.*     Reject  the  precipitate.     Treat  the  filtrate  as  follows  : 

42.  Sulfate.  Acidify  with  hydrochloric  acid.  Add  solution  of 
barium  chlorid.     Equation  : 

NajSO,        +        BaCl,        =        BaSO,        +        2NaCl 

Barium  sulfate 
(white) 

43.  Silicate.  If  silicic  acid  has  not  been  previously  detected, 
acidify  with  hydrochloric  acid,  evaporate  on  a  water  bath  to  dry- 
ness and  apply  tests  38—39  to  the  residue.  The  test  is  almost 
always  negative  (39).     Equation  :  f 

Na^SiO^     +    2HC1    =    HjSiOj     +    2NaCl 
Silicic  acid 
(white) 

44.  Chlorid.     Acidify  with  nitric  acid.     Add  solution  of  silver 

nitrate.     Equation  : 

NaCl        +        AgNOg        =        AgCl        +        XaNO, 

Silver  chlorid 
(white) 


*  A  slight  precipitate  containing  compounds  of  metals  previously  soluble  in  hot 
sodium  carbonate  solution  (37),  may  be  formed  at  this  point.  Albuminous  mat- 
ter may  also  be  present. 

t  Dried  to  a  dust  on  a  water-batb  (temperature  |  below  100°  C.)  polysilicio 
acids,  such  as  the  one  indicated  in  the  following  equation,  are  produced : 
4H,,Si03=:Il2SiA  +  3HjO.  Thorou-ihly  dried  at  temperatures  above  110°  C, 
silicic  anhydrid  results  :  HjSi^Og  =  4Si02  +  H.^0. 


18  Biochemical  Notes. 

45.  Phosphate.  Acidify  with  nitric  acid  and  apply  test  16  to 
the  remaining  portion  of  the  filtrate  (40).* 

46.  Nitrate.  Second  part  (1)  [41] .  Evaporate  to  dryness  in  a 
casserole  on  a  water  bath.  Moisten  the  residue  with  a  moderate 
excess  of  phenol  di-sulfonic  acid,t  and  return  the  mixture  to  the 
hot  water  bath.  While  the  mixture  is  being  heated,  stir  it  in  order 
to  favor  complete  disintegration  of  any  solid  organic  matter.  Cool. 
Cautiously  add  ammonium  hydroxid  in  small  quantities  until  the 
mixture  becomes  strongly  alkaline.     Filter.     Typical  equation  : 

C6H3(OH)(S03H)2  4-  SNaNOs  =  CeH^CNOJsCOH)  +  Na^SO^  +  NaHSO,  +  Hp 
Phenol  Picric  acid 

di-sulfonic  acid  (yellow) 

C6Hj(N02)3(OH)  +  NH.OH  =  C6H2(N02)30NH,  +  H^O 

Ammonium 
picrate 
(yellow) 

A  yellow  filtrate  indicates  the  previous  presence  of  nitrate.  The 
color  of  the  filtrate  is  due  to  dissolved  ammonium  picrate.  Bio- 
logical liquids  rarely  contain  nitrate  and  the  result  of  the  test  is 
usually  negative. 

47.  Sulfid.  Third  part  {\)  [41].  Acidify  with  hydrochloric 
acid.  Test  with  moist  lead  acetate  paper  any  gas  that  may  be 
formed.  Heat  the  mixture  and  continue  the  test.  The  result  is 
usually  negative.     Equations : 

Na^S      +      2HC1      =      H^S      +      2NaCl 
Hydrogen 
sulfid  (gas) 

H,S      +      PbCC^HjO,),      =      PbS      +      2C,HA 

Lead  sulfid 
(black) 

48.  Carbonate.  To  a  new  portion  of  the  original  solution  add 
hydrochloric  acid  to  acid  reaction.     If  effervescence  occurs  {a)  re- 

*To  make  certain  that  arsenic  is  not  responsible  for  the  yellow  precipitate 
that  may  occur  at  this  point  (16)  (if  a  precipitate  was  obtained  with  hydrogen 
sulfid  in  test  13),  it  is  necessary  to  acidify  the  filtrate  with  hydrochloric  acid  and 
to  treat  the  hot  solution  (70°  C. )  with  hydrogen  sulfid  gas  for  about  30  minutes 
to  precipitate  arsenic.  Filter,  expel  hydrogen  sulfid  from  the  filtrate  by  boilingi 
add  nitric  acid  and  finally  molybdie  solution.  As  a  rule  this  procedure  is  quite 
unnecessary  because  of  the  absence  of  arsenic  in  quantities  sufficient  for  detection 
(13,16). 

t  The  reagent  was  prepared  in  the  following  proportions  :  30  grams  of  phenol 
and  370  grams  of  sulfuric  acid  (1.84  sp.  gr. )  were  mixed  and  heated  at  about 
100°  C.  for  six  hours  on  a  water  bath. 


Inorganic  JSIatters.  19 

peat  the  test  with  a  sufficient  volume  of  the  liquid  in  a  closed  flask 
connected  with  lime-water  under  a  lajer  of  kerosene.  Heat  the 
acidified  mixture.  If  a  white  precipitate  forms  in  the  lime-water 
(6),  test  its  solubility  and  effervescent  properties  in  dilute  acetic 
acid  (c).     Equations  : 

a.  Na^COj     +     2HC1    =    CO^    +     NaCl     +     H^O 

Carbon  di- 
oxid  (gas) 

b.  Ca(0H)2     +     CO2    =    CaCOs     +     H^O 

Calcium 

carbonate 

(white) 

c.  CaCOs  +  2C2HPJ  =  CO2  +  CaCC^HsO^),  +  H^O 

49.  Summary  of  anion  processes,  36-48.  In  the  summary 
on  the  opposite  page  the  methods  heretofore  described  for  the  detec- 
tion of  anions  are  indicated  in  a  manner  intended  to  favor  easy 
review. 


I.  Okiginal  Solution  +  (Na)2C03,  etc.  (37).     Filter; 


Precipitate  +  HNO3,  etc.  (38).  Filteate.     Divide  into  3  parts 

Filter :  I 


s 


Kesidiie{?):     Filtrate:                        f  i  . 

Silicate  (?)        Phosphate.      Further    removal  Nitrate  (?)  Sulfia(?) 

Test  39.             Test  40.              of  metals  (?),  41.  Test  46.  Test  47. 

Filter  : 


Precipitate  :  Filtrate  : 

Reject.  A.  Sulfate,  42. 

B.  Silicate  (?),  43- 

C.  Chlorid,  44. 

D.  Phosphate,  45. 

II.  Original  Soltttion  +  HCl,  etc.  (48):  Carbonate. 


E.  Methods  for  the  Detection  of  the  Leading  Elements 
THAT  Occur  in  Biological  Organic  Compounds.*  (L  :  201- 
207.) 

50.  Carbon.  Ignite  in  an  evaporation  dish  about  10  grams  of 
pulverized  cupric  oxid,  CuO.    Cool  to  the  temperature  of  the  body 

*  Oxygen  is  not  referred  to  in  this  section  because  there  are  no  satisfactory 
methods  for  its  qualitative  determination  in  organic  compounds.  It  may  be  de- 
termined by  certain  quantitative  processes. 


20  Biochemical  Notes. 

and  then  mix  with  it  about  a  gram  of  dry  pulverized  cane  sugar, 
^i2^22^n*  Transfer  to  a  dry  hard  glass  test  tube  provided  with  a 
perforated  cork  with  L-tube  and  gradually  ignite.  Conduct  the  gas 
into  lime-water  under  a  layer  of  kerosene  (48).     Equation  : 

C12H2A1        +        24CuO        =        12C0,        +        imp        +        24Cu 

Carbon  dioxid 
(gas) 

Notice  the  metallic  copper  that  resulted.  Read  51  before  emp- 
tying the  tube. 

In  this  process  organic  carbon  is  oxidized  to  carbon  dioxid. 

51.  Hydrogen.  After  the  mixture  in  the  tube  (50)  has  been 
thoroughly  ignited,  allow  it  to  cool.  Examine  it  carefully.  Re- 
move with  a  stirring  rod  some  of  the  liquid  that  condensed  in  the 
upper  part  of  the  tube  and  bring  it  in  contact  with  anhydrous 
copper  sulfate.     Equation  : 


CuSO,        + 

oHjO 

CuSO,,  5H,0 

Cupric  sulfate 

Cupric  sulfate 

anhydrous 

hydrous 

(white) 

(blue) 

In  this  process  (50)  organic  hydrogen  is  oxidized  to  water. 

52.  Nitrogen.  Detected  as  ammonia.  To  a  small  quantity  of  dry 
pulverized  egg  albumen  in  a  mortar  add  about  ten  to  twenty  times 
the  amount  of  powdered  soda  lime.  Mix  the  two  thoroughly, 
transfer  the  mixture  to  a  dry  test  tube  and  ignite.  Apply  test 
30  to  the  gas  evolved. 

In  this  process  nitrogen  in  organic  combination  is  converted  into 
ammonia. 

53.  Detected  as  cyanid.  (Lassaigne's  test.)  Into  a  dry  test- 
tube  place  a  dry  piece  of  metallic  sodium  the  size  of  a  pea  and 
about  twice  as  much  dry  pulverized  blood.  Ignite.  Raise  the 
temperature  gradually  and  maintain  a  glow  for  a  minute  or  two. 
Dip  the  hot  end  of  the  tube  in  5  c.c.  of  water  in  an  evaporation 
dish.  The  tube  will  break  and  its  contents  will  pass  into  the 
water.  Warm  the  mixture  to  favor  solution  of  soluble  products. 
Filter  through  a  very  small  filter.  Add  several  drops  of  solutions 
of  sodium  hydroxid  and  ferrous  sulfate,  and  a  single  drop  oi  ferric 
chlorid  solution.  Boil  for  a  minute,  then  cool  the  mixture.  Fi- 
nally add  just  enough  dilute  hydrochloric  acid  to  dissolve  the  pre- 
cipitated ferrous  and  ferric  hydroxids.     The  clear  solution  becomes 


Inorganic  Matters.  21 

bluish  green.  If  the  result  is  negative  at  first  let  the  mixture 
stand.  A  precipitate  of  "  Prussian  blue  "  will  gradually  subside. 
In  this  process  nitrogen  in  organic  combination  is  converted  suc- 
cessively into  sodium  cyanid,  sodium  ferrocyauid  and  ferric  ferro- 
cyanid  ("  Prussian  blue").     See  test  2i. 

54.  Sulfur.  Into  a  dry  test  tube  place  a  dry  piece  of  metallic 
sodium  the  size  of  a  pea  and  about  twice  as  much  dry  egg  albumen. 
From  this  point  repeat  process  53  until  the  filtrate  containing  the 
soluble  products  is  obtained.  In  this  process  organic  sulfur  is  con- 
verted into  sodium  sulfid  and  the  sulfid  is  contained  in  the  filtrate. 
The  presence  of  sulfid  in  the  filtrate  may  be  determined  in  various 
ways  as  follows  (see  also  tests  59-60)  : 

55.  Sodium  nitroprussid  test.  A  few  drops  of  sodium  nitroprus- 
sid  solution  produce  violet  coloration  of  liquids  containing  alkali 
sulfids.    (L  :   181.)    The  nature  of  the  colored  product  is  unknown. 

56.  Silver  test.  Place  a  few  drops  of  the  solution  (54)  on  a  sil- 
ver coin.     A  black  stain  indicates  sulfid.     Equation  : 

2Ag      +      Na,S      +       2H,0      =      Ag,S      +      2NaOH      +      H^ 

Silver  sulfid 
(black) 

57.  In  some  organic  compounds  sulfur  \&  firmly  combined  in  the 
molecule,  in  some  it  is  loosely  united,  in  others  it  exists  in  both 
conditions  of  combination. 

58.  Tests  for  loosely  united  organic  sulfur.  Place  horn  shavings 
in  fairly  concentrated  sodium  hydroxid  solution  (10  per  cent.). 
Boil  the  mixture  several  minutes.  In  this  process  loosely  combined 
organic  sulfur  is  converted  to  sulfid.  As  a  rule  only  a  part  of  the 
total  sulfur  can  be  converted  to  sulfid.  Test  the  filtrate  with  the 
following  reagents  : 

59.  Lead  acetate.  Add  the  reagent  drop  by  drop.  Equa- 
tion (47) : 

Na,,S      +      PbCCjHjO,),      =      PbS      +      2NaCjH30j 

Lead  sulfid 
(black) 

60.  Evaporate  a  portion  nearly  to  dryness.  Acidify  with  nitric 
acid  and  test  the  gaseous  product  with  wet  lead  acetate  paper  (59). 

61.  Apply  tests  55  and  56. 

62.  Test  for  firmly  united  organic  sulfur.     Fuse  a  small  quantity 


22  Biochemical  Notes. 

of  dry  egg  albumen  with  solid  potassium  hydroxid  and  potassium 
nitrate  in  a  porcelain  crucible.  Continue  the  fusion  until  the  mass 
is  practically  white.  Cool.  Place  the  cold  crucible  and  contents 
in  a  small  beaker  with  a  little  water  and  boil  to  effect  solution. 
Acidify  with  hydrochloric  acid.  The  acid  may  be  added  before 
solution  is  complete  in  order  to  hasten  the  process.  Filter.  Add 
barium  chlorid  solution  (42). 

In  this  process  all  the  organic  sulfur  is  oxidized  and  soluble  alkali 
sulfate  results.* 

63.  Phosphorus.  Dry  a  small  piece  of  liver  in  a  watch  glass  on 
a  water  bath.  Subject  the  dry  mass  to  process  53  down  to  the  point 
of  acidifying  with  hydrochloric  acid.  In  this  case  acidify  with 
nitriG  acid  the  solution  containing  the  products  of  the  fusion  and 
apply  to  it  test  16  for  phosphate. 

In  this  process  the  organic  phosphorus  is  oxidized  and  soluble 
alkali  phosphate  results. 

64.  Iron.  Dry  a  small  amount  of  blood  in  a  crucible  on  a  water 
bath.  Add  a  few  drops  of  nitric  acid  and  ignite  cautiously,  until 
the  mass  is  charred.  Add  nitric  acid  occasionally  and  repeat  the 
ignition  process  until  incineration  is  complete.  Avoid  excessive 
heating.  See  footnote,  page  8.  Warm  the  ash  with  hydrochloric 
acid  and  to  this  solution  apply  test  21. 

In  this  process  the  organic  iron  is  oxidized,  and  converted  by 
hydrochlorid  acid  to  ferric  chlorid. 

*  In  quantitative  determinations  it  is  usually  found  that  the  sulfid  sulfur  ob- 
tainable by  process  58  is  much  less  in  quantity  than  the  sulfate  sulfur  obtainable 
by  process  62.  The  added  quantity  obtainable  by  the  latter  process  is  the  firmly 
combined  sulfur.  A  vigorous  oxidative  process  is  required  to  remove  the  firmly 
combined  sulfur  from  its  molecular  position. 


CHAPTER   II. 

FATS. 

A.     Introductory   Notes  on  Some  Solvents  of   General 
Value  in  Biochemical  Experiments. 

65.  Kinds.  In  our  study  of  the  properties  of  biological  organic 
compounds  we  shall  have  occasion  repeatedly  to  ascertain  the 
soluble  qualities  of  the  substances  under  examination.  In  order 
to  compare  the  biological  substances  with  each  other  from  the 
standpoint  of  solubility,  it  will  be  desirable  to  determine  the  effects 
of  the  same  solvents  on  the  important  compounds.  As  a  rule  we 
shall  use  two  series  of  solvents,  which  we  may  designate  as  bio- 
logical solvents  (A)  and  special  solvents  (B)  and  which  consist  of 
the  liquids  named  below  : 

A.  Biological  solvents:  Water,  saliva,  gastric  juice,  pancreatic 
juice,  blood  serum,  urine. 

B.  Special  solvents:  Sodium  chlorid  (5  f)),  hydrochloric  acid 
(0.2  fo),  sodium  carbonate  (0.5^),  concentrated  hydrochloric  acid 
(39  f)),  potassium  hydroxid  (10  ^),  alcohol  (95  ^),  ether. 

In  special  cases  additional  solvents,  such  as  bile  and  chloroform, 
will  be  employed. 

Water  and  urine  will  be  supplied  in  their  natural  conditions. 
Common  drinking  water  will  be  used.  Its  very  slight  proportion- 
ate content  of  dissolved  matters  is  without  significance  in  the  tests 
in  which  it  will  be  employed. 

Saliva,  gastric  juice,  pancreatic  juice  and  serum  cannot  be 
obtained  satisfactorily  in  sufficient  quantities  for  the  many  tests 
that  will  be  made,  but  there  will  be  available  various  artificially  p>re- 
pared  liquids  that  will  closely  resemble  the  corresponding  natural 
products  and  will  manifest  exactly  their  solvent  effects  (69-78).* 

66.  Reactions  of  the  natural  biological  solvents.  —  Of  the 

*  When  only  small  quantities  of  saliva  are  needed  the  filtered  natural  secretion 
■will  be  obtained  and  employed  by  each  student.  Normal  sf r«m  will  be  furnished 
when  only  small  quantities  are  recjuired.  In  particularly  important  connections 
solubilities  will  be  demonstrated  with  the  natural  solvents. 

23 


24  Biochemical  Notes. 

biological  solvents  water  is  neutral.  Saliva,  pancreatic  juice  and 
serum  are  slightly  alkaline,  by  reason  of  the  presence  of  salts,  such 
as  phosphates  and  carbonates,  that  undergo  hydrolytic  dissociation 
(P  :  295).  Gastric  juice  is  quite  strongly  acid  because  of  its  con- 
tent o{  free  hydrochloric  acid  and  urine  is  usually  slightly  acid  be- 
cause of  the  preponderance  of  acid  reacting  salts,  chiefly  acid  phos- 
phates (P :  295). 

67.  The  reactions  of  these  liquids  have  been  stated  in  terms  of 
their  effects  on  litmus,  an  indicator  that  is  commonly  employed, 
but  which  is  unsatisfactory  in  many  respects.  It  is  so  important 
for  us  to  clearly  understand  how  much  and  how  little  the  terms 
"  acidity  "  and  "  alkalinity  "  mean,  as  they  are  commonly  used  in 
connection  with  biological  liquids,  that  I  shall  quote  a  very  satis- 
factory statement  of  facts  in  this  connection  from  an  important 
paper  which  appeared  while  the  proofs  of  this  volume  were  being 
corrected.*  Although  the  following  statement  was  made  in  refer- 
ence to  urine,  it  applies  equally  well  to  biological  liquids  in  general 
and  expresses  the  opinion  regarding  them  in  this  connection  that 
has  been  rapidly  gaining  acceptance  for  several  years : 

"A  large  number  of  methods  have  been  published  for  determin- 
ing and  estimating  the  reaction  of  the  urine.  .  .  .  The  figures  vary 
enormously  according  to  the  method  and  the  indicator  used  for  the 
purpose. 

"  In  addition  to  the  experimental  difficulties  introduced  by  the 
fact  that  the  urine  is  itself  colored,  and  hence  interferes  with  the  deli- 
cacy of  the  reaction  to  colored  indicators,  there  is  the  more  impor- 
tant fact  that  the  reaction  of  the  urine  is  never  due  to  free  acid  or 
to  free  alkali,  but  to  a  varying  mixture  of  salts  such  as  the  primary 
and  secondary  phosphates  of  the  alkalies.  In  such  a  mixture  the 
urine  reacts  entirely  differently  to  different  indicators.  Thus  the 
same  sample  of  normal  urine  is  acid  to  a  sensitive  indicator  to 
weak  acids  such  as  phenol-phthalein  and  alkaline  to  a  stable  indi- 
cator such  as  methyl-orange  or  di-methyl-amido-azo-benzol.  Also 
if  the  titration  figures  to  three  such  indicators  as  phenol-phthalein, 

*  Edward  S.  Edie  and  Edward  Whitley.  A  method  for  determining  the  total 
daily  gain  or  loss  of  fixed  alkali  and  for  estimating  the  daily  output  of  organic 
acids  in  the  urine,  with  applications  in  the  case  of  Diabetes  melHtus.  The  Bio- 
Chemical  Journal,  January,  1906  ;  vol.  I,  pages  11,  12  and  13. 


Fats.  25 

litmus,  and  ^  di-methyl '  be  taken,  it  will  be  found  that  there  is  a 
high  acidity  with  the  phenol-phthalein,  a  much  lower  acidity  with 
the  litmus,  and  a  high  alkalinity  with  the  di-methyl  indicator. 
AVhen  similar  titrations  are  undertaken  in  the  case  of  an  artificial 
mixture  of  the  primary  (NaH2P0J  and  secondary  (Na2HPOJ 
phosphates  of  sodium  in  water  to  which  such  phosphates  have  been 
added  in  known  amount  and  ratio,  the  reason  underlying  the  dif- 
ferences in  the  titration  values  becomes  at  once  apparent.  The 
neutral  point  for  phenol-phthalein  lies  at  the  point  where  the 
kations  and  anions  are  so  distributed  as  to  correspond  to  NagHPO^, 
while  for  di-methyl  (or  methyl  orange)  the  neutral  point  corresponds 
to  NaH2PO^,  and  for  litmus  the  neutral  point  lies  somewhere  inter- 
mediate between  these  two  values.  It  is  clear  from  this  statement 
that  it  is  futile  to  regard  any  one  indicator  as  the  arbiter  of  neu- 
trality, and  to  consider  a  solution  as  being  neutral  because  it  is 
neutral  to  phenol-phthalein  when  it  is  alkaline  to  litmus  and  '  di- 
methyl,' or  when  it  is  neutral  to  litmus  or  lacmoid,  and  acid  to 
phenol-phthalein  and  alkaline  to  di-methyl  at  the  same  time. 

"  The  only  true  definition  of  neutrality  would  be  the  point  at  which 
the  concentrations  of  hydrogen  and  hydroxyl  ions  are  equal,  and  no 
colored  indicator  satisfies  this  condition  but  indicates  neidrality  at  a 
point  tohere  there  is  a  given  ratio  other  than  equality  between  the  acidic 
and  basic  ions.  The  value  of  this  ratio  depends  on  the  ease  with 
which  the  colored  indicator,  and  its  ions,  associate  or  dissociate  in  solu- 
tion. 

"  The  proper  method  of  determining  reaction  ought,  therefore,  to 
be  some  method  of  determination  of  the  concentration  of  hydrogen 
or  hydroxyl  ions  which  would  indicate  where  these  two  concentra- 
tions were  equal. 

"  In  the  case  of  a  solution  of  the  mixed  phosphates,  such  as  the 
urine,  all  physical  methods  for  determination  of  the  ionic  concen- 
trations fail,  however,  in  accuracy  on  account  of  the  very  slow 
variation  of  the  concentration  in  the  two  ions  around  the  neutral 
point.  In  the  case  of  free  acid  or  free  alkali,  the  smallest  addition 
of  acid  or  alkali  to  the  solution  in  the  neighborhood  of  the  neutral 
point  causes  an  immense  swing  in  the  ratio  of  the  two  ions  which 
is  at  once  obvious  in  the  gas  battery ;  whereas,  in  the  case  of  a 
solution  containing  phosphates,  the  degree  of  dissociation  is  low, 


26  Biochemical  I^otes. 

and  an  addition  of  acid  or  alkali  causes  not  a  great  swing  in  the 
ratio  of  hydrogen  or  hydroxyl  ions  but  a  decomposition  of  phos- 
phate in  either  direction,  and  the  establishment  of  a  new  equilibrium 
in  which  the  ratio  of  the  two  ions  may  not  be  widely  different  from 
the  former. 

"  On  this  account  a  solution  such  as  the  urine,  or  any  of  the  body 
fluids  in  general,  behaves  as  a  controlling  agent  or  as  a  neutralizing 
agent  for  either  acid  or  alkali,  and  prevents  large  variations  in 
either  hydrogen  or  hydroxyl  ion  concentration. 

"  The  importance  of  such  a  regulating  mechanism  to  the  organism, 
the  cells  of  which  are  so  sensitive  to  such  variations,  is  too  obvious 
to  need  elaboration." 

68.  Digestive  powers  of  the  natural  biological  solvents. 
Saliva,  gastric  juice  and  pancreatic  juice  are  dilute  aqueous  solu- 
tions and  each  contains  enzymes  that  transform  Ghemically  certain 
organic  substances.  Saliva  is  especially  active  in  transforming 
(digesting)  complex  carbohydrates,  like  starch,  into  simpler  carbo- 
hydrates like  maltose.  Gastric  juice  vigorously  converts  (digests) 
complex  proteins,  like  globulin,  into  simpler  proteins  like  globu- 
loses.  Pancreatic  juice  accomplishes  all  in  a  digestive  way  that  both 
saliva  and  gastric  juice  effect  on  the  two  classes  of  substances  indi- 
cated, and,  besides,  it  hydrates  fats  into  glycerol  and  fatty  acids  (82). 

Water  is  free  from  enzymes.  Serum  and  urine  do  not  manifest 
digestive  effects  with  suificient  distinctness  to  require  attention  in 
this  regard.  Serum  is  a  dilute  alkaline  aqueous  solution  containing 
especially  proteins  and  saline  matter.  Urine  is,  in  the  main,  a 
dilute  aqueous  saline  solution,  containing  especially  chlorids,  phos- 
phates and  sulfates,  and  several  organic  substances,  among  which 
urea  (L  :  251)  is  the  most  conspicuous. 

General  composition  of  the  natural  biological  solvents.  69. 
Saliva  (mixed)  is  a  very  slightly  alkaline  solution  (67)  containing 
about  0.3  per  cent,  of  protein  and  other  organic  matter ;  also  ap- 
proximately 0.2  per  cent,  of  inorganic  compounds,  consisting  chiefly 
of  phosphate,  carbonate,  chlorid  and  sulfate  of  potassium  and 
sodium.  It  contains  traces  of  enzymes,  chief  of  which  is  ptyalin, 
which  hydrates  polysaccharid  to  disaccharid.  The  alkalinity  is 
usually  not  greater  than  that  of  a  0.15  per  cent,  to  0.2  per  cent, 
solution  of  sodium  carbonate. 


Fats.  27 

The  composition  of  the  artificial  saliva  prepared  for  use  in  the 
tests  (65)  is  indicated  in  section  75. 

70.  Gastric  juice  is  a  decidedly  acid  solution  containing  about 
0.3  per  cent,  of  protein  and  other  organic  matters  and  approximately 
0.4  per  cent,  of  inorganic  substances.  Of  these,  chlorids  of  sodium 
and  potassium,  and  hydrochloric  acid,  are  the  most  conspicuous. 
It  contains  slight  quantities  of  enzymes,  chief  of  which  is  pepsin, 
which  hydrates  complex  proteins  like  albumin  to  simpler  proteins 
like  peptone.  The  acidity  is  commonly  equal  to  that  of  a  0.2  per 
cent,  solution  of  hydrochloric  acid. 

The  composition  of  the  artificial  gastric  juice  prepared  for  use  in 
the  tests  (65)  is  indicated  in  section  76. 

71.  Pancreatic  juice  is  a  slightly  alkaline  liquid  containing  about 
10  per  cent,  of  solids,  of  which  9  per  cent,  consists  of  organic  mat- 
ter, chiefly  proteins.  Alkali  chlorids,  phosphates  and  carbonates  are 
the  chief  inorganic  constituents.  It  contains  enzymes  similar  to 
those  in  saliva  and  gastric  juice  and  also  lipase,  2l  fat-splitting  enzyme, 
in  very  active  proportion.  The  alkalinity  is  approximately  equal 
to  that  of  a  0.25  per  cent,  solution  of  sodium  carbonate. 

The  composition  of  the  artificial  pancreatic  juice  prepared  for 
use  in  the  tests  (65)  is  indicated  in  section  77. 

72.  Serum  is  a  slightly  alkaline  liquid  containing  about  9  per 
cent,  of  solid  matter,  of  which  about  1  per  cent,  consists  of  inor- 
ganic substances.  The  organic  matter  consists  chiefly  of  proteins 
and  "  extractives."  The  inorganic  matter  is  mainly  sodium  chlorid, 
together  v/ith  slight  proportions  of  the  usual  salts  present  in  biolog- 
ical liquids.  The  alkalinity  of  the  serum  is  not  greater  than  that 
of  a  0.25  per  cent,  solution  of  sodium  carbonate. 

The  composition  of  the  artificial  serum  for  use  in  the  tests  (65) 
is  indicated  in  section  78. 

73.  Ui'ine  is  a  dilute  saline  solution  that  varies  considerably  in 
reaction  but  which  is  more  frequently  acid  than  alkaline.  The 
acidity  is  probably  never  greater  than  that  of  a  0.1  per  cent,  solu- 
tion of  hydrochloric  acid.  The  urine  usually  contains  about  3  to  5 
per  cent,  of  dissolved  substances,  although  the  proportion  of  soluble 
matter  varies  considerably.  The  reaction  is  due  to  hydrolytically 
dissociated  salts,  such  as  phosphates  (67).  The  proportions  of  total 
organic  and  total  inorganic  matters  are  about  the  same.     The  chief 


28  Biochemical  Notes. 

organic  substance  is  urea,  which  amounts  to  very  much  more  than 
all  the  other  organic  substances  combined.  Sodium  chlorid  often 
predominates  quantitatively  over  all  the  other  inorganic  substances 
combined. 

The  general  composition  of  normal  urine  is  indicated  on  the 
opposite  page. 

74.  Approximate  composition  of  the  artificially  prepared  bio- 
logical liquids  to  be  used  in  the  tests  of  solubilities.  In  the 
artificial  preparation  of  the  several  "  biological "  solvents  already 
referred  to  (65)  no  attempt  has  been  made  to  represent  all  the  known 
constituents  of  the  natural  products.  It  has  been  considered  suffi- 
cient for  our  purposes  to  make  up  aqueous  solutions  containing 
constituents  approximately  identical  in  quality  and  quantity  with 
those  occurring  in  largest  proportions  in  each  liquid  under  natural 
conditions^  and  to  make  composite  solutions  that  may  be  relied  upon 
to  show  essentially  the  same  solvent  effiscts  as  the  corresponding 
natural  solutions.  It  should  be  understood  also  that  even  under 
perfectly  normal  conditions  the  natural  products  vary  considerably 
in  composition,  the  urine  especially.  Our  artificial  products  will 
show  us  the  effects  that  are  usually  manifested  by  the  natural  solu- 
tions.    Their  composition  is  given  below. 

75.  Artificial  saliva.  76.  Artificial  gastric  juice. 

Per  cent.  Per  cent. 

Albumin 0.30  Albumin 0.30 

Diastase  (commercial) 0.10  HCl 0.20 

KjHPOi 0.10  Pepsin  (commercial) 0.10 

KCl 0.05  NaCl 0.10 

CaCl2 0.03  CaCl 0.05 

NajSOi 0.03  KHjPOi 0.05 

NaHCOs 0.02 

KSCN 0.01 

77.  Artificial  pancreatic  juice.  78.  Artificial  hlood  serum. 

Per  cent.  Per  cent. 

Albumin 8.0        Albumin 8.00 

NaCl 0.6        NaCl 0.70 

NaHCOs 0.2        Meat  extract  (commercial) 0.10 

K3HPO1 0.2        KCl 0.08 

Pancreatin  (commercial) 0.2        Na2HP0^ 0.04 

Diastase  (commercial) 0.2        NaHCOg 0.02 

Lipase  (slightly  alkaline  glycerol  CaClj 0.02 

extract  of  pancreas) 0.2        MgSOi 0.01 


Fats.  29 

7g.   Urine  {normal). 

(Figures  for  daily  eliminations  —  periods  of  24  hours.) 

Grams. 

Water 1,200-1,700 

Solids  60-70 


Inorganic  solids 

. 

Org 

ajiic 

solids  : 

25-35  grams. 

25- 

35  grams. 

Grams. 

Grams. 

NaCl 

.10-20 

Urea.. 

..20-30 

Na    ^ 

K 

(■SO,  1 

Urate 

1 

NH, 

L.jpo, 

►10-15 

Creatinin 

}■    3-5 

Ca 

IcOs  J 

Hippurate, 

etc. 

J 

Mg  J 

B.  Demonstration  of  Fatty  Products. 

80.  Samples  of  typical  fats,  oils,  waxes,  lecithin,  fatty 
acids,  soaps  and  glycerol. 

C.     Chemical  Constitution  of  Fats  (0  :  163). 

81.  Fats  are  glyceryl  esters  of  fatty  acids,*  They  occur  in 
both  plants  and  animals,  frequently  in  large  proportions.  Their  con- 
stituent radicals  are  indicated  by  the  following  equation  : 


C„H..COO|H      HO^CH.         C.,H„COO-CH. 
C17H35COO  i  H  +  HO  :  CH     =  C17HS5COO  —  CH  +  3H,0 

C17H35COO  i  H HO  J  CH,  C17H35COO  —  CH, 

Stearic  acid  Glycerol  Tristearin 

(three  molecules)    (one  molecule)  (one  molecule) 

82.  Formulas  of  typical  fats  f  and  the  corresponding  acids 
that  may  be  derived  from  them  by  direct  cleavage  (saponifica- 
tion, 119-126). 

*  A  few  fats  contain  radicals  of  oxy-fatty  acids,  such  as  monoxy-steario  acid, 
and  also  of  acids  that  are  not  members  of  the  common  fatty  acid  series,  as  in  the 
case  of  triolein  (0  :  163),  which  contains  the  residue  of  oleic  acid  (82)  the  eigh- 
teenth member  of  the  acrylic  acid  series.  The  members  of  the  acrylic  acid  series 
are  unsaturated  fatty  acids. 

t  The  fats  are  triglycerids.  The  more  common  fats  are  simple  triglycerids  and 
consist  of  glyceryl  combined  with  tJiree  fatty  acid  residues  of  the  same  kind.  Thus, 
tripalmitin  contains  three  palmityl  radicals.  But  most  animal  and  vegetable 
fatty  deposits  also  contain  mixed  triglycerids,  in  one  type  of  which  only  iico  of  the 
acid  radicals  are  the  same  and  in  the  other  type  the  three  acid  radicals  are  different. 


30 


Biochemical  Notes. 


Fats  : 

CjH^COO  — CH2  CigHajCOO  — CHj  Ci^HgsCGO  —  CH,  C17H33COO  —  CH„ 

I  I  I  I 

CsH^COO  — CH  CisHaiCGO  — CH  C17H35COO  — CH  CiTHasCOO  — CH 


C3H7COO  — CHj 

(CisHjeOg) 
Tributyrin 

Fatty  acids 
C3H7COOH 

Butyric  acid 


C15H31COO— CH2 

(CsiHggOe) 
Tripalmitin 


C15H3JCOOH 

Palmitic  acid 


CnHssCOO  — CHj 

(CstHuoOb) 
Tristearin 


C„H35COOH 

Stearic  acid 


Ci7H33COO  —  CH2 

(CsrHioiOe) 
Triolein 


C^HssCOOH 

Oleic  acid 


The  unsaturated  character  of  oleic  acid  and  its  close  relationship 
to  palmitic  acid  and  to  stearic  acid  are  shown  by  the  following 
equations,  which  express  oxidation  effects  of  fusion  with  potassium 
hydroxid,  and  a  reduction  effect  of  treatment  with  phosphorus  and 
iodin  : 

Oxidation:  C17H33COOH  +  2K0H  =  C15H31COOK  +  CK3COOK  +  Hj 
Oleic  acid  Potassium  Potassium 

palmitate  acetate 


Beduction . 


C17H33COOH  +  H2  =  C17H35COOH 

Stearic  acid 


D.     Geneeal  Characters  of  Common  Crude  Animal 
AND  Vegetable  Fats.  * 

Typical  fats  for  use  in  the  tests  to  be  described.  83.  Kinds. 
In  experiments  86-161  use  samples  of  one  or  more  of  the  following 
common  crude  animal  and  vegetable  fatty  products  :  tallows  (beef 
and  mutton),  lard,  butter,  olive  oil  and  myrtle  wax. 

84.  Nature.  Each  of  these  crwcZe  fatty  materials  consists  of  a 
mixture  of  fats,  and  also  contains  water  together  with  slight  quanti- 
ties of  unimportant  inorganic  and  organic  matters.     Myrtle  wax  is 

*  Fat  is  a  term  commonly  applied  to  any  mass  of  fat  or  fats,  pure  or  crude, 
such  as  butter.     A  fat  is  a  particular  chemical  substance  of  definite  chemical  and " 
physical  properties,  such  as  tripalmitin  {82). 

Some  fats  and  waxes  are  physically  very  much  alike.  Fats  are  esters  of  a  par- 
ticular tri-hydric  alcohol  of  relatively  low  moleculai  weight,  i.  e.,  glycerol,  whereas 
waxes  are  esters  of  various  mono-hydric  alcohols  of  relatively  high  molecular  weight, 
e.  g.,  cetyl  palmitate  (in  spermaceti),  C15H31COO  —  C15H35.  Wax,  like  fat,  is  a 
crude  mixture  (0  :  162-163). 

The  terms  wax  and  fat  are  frequently  used  interchangeably. 

Oils  are  of  three  general  kinds  :  (1)  Fatty  or  fixed  oils,  such  as  olive  oil  ;  (2) 
essential  or  volatile  oils,  such  as  oil  of  turpentine  ;  (3)  mineral  oils,  such  as  kero- 
sene. Fatty  oils  are  of  both  animal  and  vegetable  origin,  and  are  the  only  oils 
that  contain  true  fats.  Fatty  oil  is  physically  unlike  fat  in  being  liquid  at 
room  temperature.     Qualitatively  fats  and  fatty  oils  are  similar  (0  :  162). 


Fats.  31 

somewhat  exceptional  (135).     The  impurities  referred  to  exert  no 
appreciable  influences  in  the  tests  to  be  employed. 

85.  Preparation.  Each  kind  of  animal  fat  here  referred  to  is 
liquid  at  body  temperature  but  is  solid  at  room  temperature.  For 
the  preparation  of  the  first  three  varieties  the  fatty  tissue  of  the 
animal  is  finely  divided  and  heated,  when  the  fat  may  be  expressed 
in  molten  condition.     It  quickly  solidifies. 

Butter  is  made  from  milk  by  a  churning  process  that  brings  the 
suspended  fat  globules  together  in  buttery  masses  of  irregular  size 
which  may  be  easily  pressed  together. 

Olive  oil  is  obtained  from  the  fruit  of  the  common  olive,  chiefly 
by  simple  expression. 

The  berries  of  the  wax-myrtle  (llyrica  ccrifera)  are  coated  with  a 
fatty  layer  which  is  commonly  called  ''  myrtle  wax,"  or  "  myrica 
tallow."  The  wax-myrtle  and  its  fruit  are  also  designated  by  the 
term  bay  berry  and  myrtle  wax  is,  therefore,  frequently  called 
*'  bayberry  tallow."  * 

E.     Elementaey  Composition  of  Fats. 

86.  True  fats  consist  solely  of  carbon,  hydrogen  and  oxygen. 
Apply  tests  (50-51)  to  a  sample  of  one  of  the  fatty  products.  Prove 
the  absence  of  nitrogen,  sulfur,  phosphorus  and  iron,  tests  53,  62, 
63,  64. 

F.     Physical  Propeeties  of  Fats. 

87.  Fats  are  non-volatile  (footnote,  page  30).  Notice  the 
greasy  stains  made  on  paper  by  soft  fats,  such  as  lard  and  olive 
oil.  Place  the  spotted  paper  on  a  watch  glass  and  heat  without 
burning. 

88.  Pure  fats  are  substances  of  neutral  reaction.  —  Test  with 
litmus  the  reaction  of  fresh  butter  and  /"resh  olive  oil. 

89.  Rancid  fat  is  acid  in  reaction.  —  Apply  test  88  to  old  but- 
ter and  old  olive  oil.  The  acid  in  rancid  fat  results  from  hy- 
drolysis of  fat  molecules  (especially  through  the  agency  of  bacteria), 
as  shown  by  the  typical  equation  on  the  next  page  : 

*The  fruit  of  the  common  bay  tree  oi-  laurel  {Laurus  nobilis)  is  also  termed 
bayberry.  (Bay  rum  is  obtained  by  distilling  with  rum  the  leaves  of  Pimenta 
acrin,  one  of  the  members  of  the  myrtle  family,  or  by  mixing  with  alcohol,  water 
and  acetic  ether,  the  volatile  oil  obtained  from  the  leaves  by  distillation.') 


32  Biochemical  Notes. 

C3H,C00— CHj  CH2OH 

I  I 

CgH,COO— CH    +  3H0H  =  3C3H7COOH  +  CHOH 

C3H7COO— CHj  CH2OH 

Tributyrin  Butyric  acid  Glycerol 

90.  Solubility.  Test  the  solubility  of  one  of  the  fats  in  all  the 
solvents  referred  to  in  section  65. 

91.  Pour  upon  a  piece  of  filter  paper  a  drop  of  any  of  the  true 
solutions  obtained,  e.  g.y  the  ethereal  solution  (90).  Notice  the  greasy 
stain  (87)  that  is  produced  by  evaporation  of  the  solvent. 

92.  Allow  the  true  solutions  (90)  to  evaporate  spontaneously  in 
test  tubes.  Examine  under  the  microscope  the  deposits  produced. 
See  experiment  loi. 

93.  Emulsion.  Shake  vigorously  in  a  test  tube  a  mixture  of 
about  5  c.c.  of  water  and  2  or  3  drops  of  fresh  perfectly  neutral 
olive  oil  (89).  Note  the  time  at  which  the  experiment  was  begun, 
and  put  the  tube  in  a  water  bath  at  about  40°  C.  Ten  minutes 
after  the  beginning  of  the  experiment  examine  the  liquid  under  the 
microscope.    Acidify  with  hydrochloric  acid. 

94.  Repeat  experiment  93  but  use  5  c.c.  of  a  soap  solution  instead 
of  water.  After  microscopic  examination,  acidify  with  hydrochloric 
acid  and  compare  with  the  result  in  test  93. 

95.  Repeat  experiment  93  but  use  5  c.c.  of  0.5  per  cent,  sodium 
carbonate  or  hydroxid  solution  instead  of  water.  After  microscopic 
examination  of  the  mixture  add  a  drop  of  oleic  acid.  Shake  the 
mixture.     Repeat  the  latter  part  of  experiment  94. 

96.  Examine  milk  under  the  microscope  and  compare  with  the 
microscopic  observations  in  experiments  93-95.  Acidify  the  milk 
as  in  experiments  93-95. 

97.  Let  a  drop  of  perfectly  neutral  olive  oil  or  melted  fresh 
butter  fall  gently  upon  the  surface  of  0.25  per  cent,  sodium  car- 
bonate solution  contained  in  a  watch  glass.  Notice  that  the  film  of 
oil  on  the  surface  remains  clear.  Compare  with  the  results  of  ex- 
periments 98  and  99. 

98.  Add  2  drops  of  oleic  acid  to  5  c.c.  of  neutral  olive  oil  or 
melted  fresh  butter  in  a  dry  test  tube.  Shake  vigorously.  With 
a  drop  of  this  solution  repeat  the  preceding  experiment  (97)  in 
another  watch  glass.  Notice  the  diffusion  currents  shown  by  the 
amoeboid  movement  of  the  rancid  oil.     Observe  also  the  spontane- 


Fats.  33 

ous  emulsiou  of  the  fatty  material.     Compare  with  the  results  of 
experiment  97. 

99.  Treat  the  remainder  of  the  rancid  oil  prepared  in  the  pre- 
ceding experiment  (98)  with  10  more  drops  of  oleic  acid.  Shake 
vigorously.  Repeat  experiment  98  with  a  drop  of  this  relatively 
very  acid  oil.  Notice  that  the  drop  of  oil  becomes  white  and 
opaque,  but  that  neither  the  general  amoeboid  movement  nor  emul- 
sion occurs. 

The  presence  of  a  large  proportion  of  oleic  acid  in  the  oil  results 
in  the  production  of  an  opaque  soapy  coating  on  the  oil.  The  caked 
soapy  coating  is  relatively  insoluble,  and  mechanically  prevents  the 
formation  of  an  emulsion  as  well  as  the  disintegration  of  the  main 
mass  of  the  oil. 

100.  Crystallinity.  Examine  various  samples  of  fat  under 
the  microscope. 

lOi.  Heat  a  small  quantity  of  fat  with  just  enough  alcohol  to 
dissolve  it.  Let  the  mixture  cool.  Examine  the  deposit.  See 
experiment  92. 

Demonstrations.  102.  Influence  of  albuminous  compounds 
and  other  substances  on  emulsion.  103.  Preparation  of  a  pure 
fat.  104.  Specific  gravity  of  fats.  105.  Effects  of  lowering 
the  temperature  of  fatty  oils.  106.  Effects  of  raising  the  tem- 
perature of  solid  fats  and  waxes.  107.  Inflammability  of  fats. 
108.  Decomposition  of  fats  by  destructive  distillation,  with  an 
examination  of  the  products.  109.  Apparatus  for  determining 
the  melting  point  of  a  substance. 

no.  Determination  of  melting  point.  —  Determine  by  the 
method  shown  in  the  demonstration  (109),  the  melting  point  of  any 
of  the  solid  fats. 

III.  Table  showing  the  melting  points  of  typical  pure  fats 
and  of  the  corresponding  acids  that  may  be  derived  from  them 
by  direct  cleavage  (saponification,  1 19-126). 


Formula. 

Melting  point. 

Fat. 

Fatty  acid. 

Fat. 

°  C. 

Fatty  acid 

Tributyriu 

CisHjsOg 

n-C3H,C00H 

9 

—  8 

Tripalinitin 

CaiHggOg 

C,5H3iCOOH 

50 

63 

Tristearin 

t'57  "hqOg 

CnHgsCOOH 

57 

70 

Triolein 

CoiHioiOe 

CnHooCOOH 

—  6 

14 

34  Biochemical  Notes. 

G.     Chemical  Tests. 

112.  Acrolein  test.  —  Heat  a  small  quantity  of  fat  in  a  dry  test 
tube.  Pungent  fumes  of  acrolein  *  will  be  eliminated.  Make  a  few 
drops  of  silver  nitrate  solution,  on  a  watch  glass,  alkaline  with  a 
drop  of  ammonium  hydroxid.  Note  the  reducing  effect  of  the 
acrolein  fumes  on  some  of  this  solution  in  a  strip  of  filter  paper 
suspended  directly  over  the  mouth  of  the  test  tube. 

The  characteristic  odor  of  burning  fat  is  partly  due  to  the  acro- 
lein that  is  formed  in  the  process.  The  maximum  production  of 
acrolein  requires  the  presence  of  a  special  dehydrating  agent.  Try 
the  effect  of  such  an  agent  in  the  next  test. 

113.  Repeat  the  preceding  test,  but  before  heating  the  fat  mix 
with  it  about  twice  its  quantity  of  dry  potassium  hydrogen  sulfate, 
which  effects  dehydration. 

Under  the  conditions  of  the  acrolein  test  fat  is  decomposed  into 
glycerol  and  other  products.  The  glycerol  is  dehydrated  into 
acrolein.     See  test  112.     Equation: 

CH2OH  CHO 

I  I 

CHOH         =        CH         +        2H2O 

I  II 

CH2OH  CH2 

Glycerol  Acroleinf 

(Acrylic  aldehyde) 

*  The  name  is  derived  from  acer,  sharp,  and  oleum,  oil. 

t  Acrolein  ia  the  aldehyde  of  allyl  alcohol.  The  corresponding  acid  is  acrylic 
acid  (81).  Both  compounds  are  ethylene  (HjC:=CH3)  or  olefin  derivatives  and 
are  typical  unsaturated  compounds  (82).  Their  relations  to  each  other  are  indi- 
cated by  the  following  formulas  : 

CHjOH  iH^O        CHO  COOH 

CH  +0         ss-^        CH     +     O     &^    CH 

II  II  II 

CH2  CH2  Ciij 

Allyl  alcohol  Acrolein  Acrylic  acid 

These  three  products  are  the  first  members  of  corresponding  homologous  series. 
Oleic  acid  (81)  is  the  eighteenth  member  of  the  acrylic  acid  series.  The  reduc- 
ing action  shov?n  by  acrolein  (112)  might  be  inferred  from  its  aldehyde  character 
(L:  226). 

In  this  connection  it  may  be  noted  that  unsaturated  compounds,  such  as  oleic 
acid,  tend  under  favorable  conditions  to  undergo  conversion  into  saturated  sub- 
stances, t.  c,  double  bonds  between  carbon  atoms  become  single  bonds  (82).  In 
such  a  conversion,  the  substances  may  exert  reducing  action.  Thus,  fat  contain- 
ing a  radical  of  any  member  of  the  acrylic  acid  series,  such  e^s  the  radical  of  oleic 


Fats.  35 

All  fats,  glycerol  and  glyceryl-containing  substances,  such  as 
lecithin  (0 :  164),  respond  to  the  acrolein  test.  Allyl  alcohol  may 
be  readily  oxidized  to  acrolein,  its  aldehyde  (see  footnote,  page  34). 

Demonstrations.  114.  Osmic  acid  and  Sudan  III  tests  for 
fat  applied  to  olein,  palmitin,  stearin  and  crude  fats.  115. 
Affinity  of  fat  for  iodin.  116.  Methods  and  apparatus  for  the 
quantitative  determination  of  fat. 

117.  Percentage  amounts  of  fat  (ether-soluble  matter)  ob- 
tained from  some  mammalian  parts.* 

The  figures  in  the  subjoined  table  represent  general  average  con- 
tents of  fatty  matters  in  the  materials  named  : 

Sweat  0.001  Bone  (shaft) 1.4 

Vitreous  humour 0.002  Bile 1.4 

Saliva  0.02  Crystalline  lens 2.0 

Lymph 0.05  Liver 2.4 

Synovia 0.06  Muscle  3.3 

Liquor  amnii 0.06  Hair 4.2 

Chyle 0.2  Milk 4.3 

Mucus 0.3  Brain 8.0 

Blood 0.4  Nerves 22.1 

Ligament  and  tendon 1.1  Adipose  tissue 94.6 

Cartilage 1.3  Bone  Marrow 96.0 

The  characters  of  the  fatty  matters  in  the  parts  indicated  above 
will  be  considered  during  the  study  of  the  tissues,  secretions,  etc. 

H.     Saponification  (0  :  147). 

Demonstrations.  118.  Various  soaps  and  their  properties. 
119.  Preparation  of  lead  oleate  ("lead  plaster").  120.  Sa- 
ponification in  distilled  water.  121.  Saponification  in  dilute 
acid.  122.  Saponification  through  the  agency  of  bacteria. 
123.  Saponification  by  lipase.  124.  Separation  of  fat  from 
soaps  and  fatty  acids.  125.  Effects  of  fatty  acids  on  carbon- 
ates. 

126.  Saponification  of  olive  oil.  To  about  10  c.c.  of  olive  oil 
or  the  same  bulk  of  any  fat  in  a  casserole  add  30  c.c.  of  sodium 
hydroxid  solution  (10  per  cent.).     Boil  the  mixture  until  it  appears 

acid  (oleyl),  blackens  osmic  acid  (demonstration  114)  by  reducing  the  latter  to  a 
lower  oxid  (black).  Fats  like  tripalmitin  and  tristearin,  which  contain  only 
radicals  of  saturated  compounds,  do  not  blacken  osmic  acid. 

*The  proportion  of  fat  in  some  of  the  above-named  parts  varies  greatly. 


36  Biochemical  Notes. 

to  be  homogeneous  and  no  oil  separates  on  transferring  several 
drops  of  it  to  water  in  a  test  tube.  Fifteen  minutes  or  more  may  be 
required  to  complete  the  treatment.  Add  water  occasionally  as 
evaporation  goes  on  in  order  to  maintain  approximately  the  original 
volume.  In  this  process  the  fat  is  completely  saponified.  Keep 
the  mixture  (127-134).     Typical  equation  : 

CnHssCOO— CH2  CH2OH 

C17H33COO— CH  +  SNaOH  =  SCnHssCOONa  +  CHOH 

Ci^HssCOO— CH2  CHjOH 

Triolein  Sodium  Glycerol 

oleate  (soap) 

127.  Frothiness  of  the  soap  solution  (137).  Notice  the  frothi- 
ness  of  a  sample  of  the  soap  solution  just  prepared  (126).  Com- 
pare with  the  frothiness  of  a  solution  of  a  piece  of  common  soap. 

128.  Emulsifying  power  of  the  soap  solution  (149).  Add  to 
a  sample  of  the  soap  solution  prepared  in  process  126,  a  few  drops 
of  olive  oil.  Compare  the  emulsion  eifects  with  those  obtained  in 
experiments  93-99. 

129.  Process  of  '*  salting  out "  the  soap.  To  a  sample  of 
the  soap  solution  (126)  add  small  quantities  of  finely  powdered 
sodium  chlorid  and  stir  thoroughly  after  each  addition.  Notice 
the  precipitate  that  rises  to  the  surface  of  the  mixture.  Filter  the 
mixture.  Make  a  watery  solution  of  the  precipitate  and  repeat 
with  it  experiments  127  and  128. 

This  precipitation  method  is  commonly  employed  in  the  process 
of  manufacturing  solid  soaps.  The  excess  of  sodium  ions  in  the 
solution  that  is  introduced  by  the  addition  of  the  sodium  chlorid, 
decreases  the  solubility  of  the  sodium  soaps  present  in  the  solution 
until  they  are  precipitated.     The  change  is  only  a  physical  one. 

130.  Conversion  of  the  sodium  soaps  into  soaps  of  other  metals. 
To  a  sample  of  the  soap  solution  (126)  add  calcium  chlorid  solution. 
An  insoluble  calcium  soap  is  formed.  Calcium  soaps  are  formed  when 
soluble  soaps  are  dissolved  in  "  hard  "  water.     Typical  equation  : 

2C„H33COONa  +  CaCl^  =  (Ci7H33COO)2Ca  +  2NaCI 

Calcium  oleate 

131.  Repeat  experiment  130  with  solutions  of  salts  of  heavy 
metals,  such  as  lead  acetate,  ferric  chlorid  and  copper  sulfate. 


Fats.  37 

132.  Decomposition  of  sodium  soap  into  fatty  acid  and  com- 
mon salt.  To  the  remainder  of  the  soap  solution  prepared  in  pro- 
cess 126  add  sufficient  hydrochloric  acid  to  make  the  mixture  acid. 
Fatty  acids  rise  to  the  top  of  the  mixture.     Typical  equation  : 

Ci,H33COONa  +  HCl  —  C17H33COOH  +  NaCl 

133.  Conversion  of  fatty  acid  into  soap  (148).  Transfer  the 
mixture  (132)  to  a  wet  filter,  which  will  retain  the  fatty  acids. 
Carefully  neutralize  the  filtrate  with  sodium  hydroxid  and  retain  it 
for  use  in  test  161 .  Remove  the  mixture  of  fatty  acids  from  the 
filter.  Add  to  it  sodium  hydroxid  and  convert  the  acids  into  soaps 
with  the  aid  of  heat. 

134.  To  the  solution  just  prepared  apply  tests  127  and  128. 

I.     Preparation  of  a  Single  Fatty  Acid  in  Compara- 
tively Pure  Condition. 

135.  Myrtle  wax  (85)  consists  chiefly  of  palmitic  acid.  About 
one  fifth  of  the  material  is  tripalmitin,  and  approximately  four 
fifths  of  it  is  free  palmitic  acid.  The  material  also  contains  a  trivial 
proportion  of  lauric  acid,  CjjHjgCOOH,  either  free  or  as  trilaurin 
or  perhaps  in  both  forms. 

Prepare  palmitic  acid  from  myrtle  wax  by  the  following  process 
136-142. 

136.  Saponification.  Place  10  grams  of  myrtle  wax  *  in  150 
c.c.  of  water  contained  in  a  small  casserole.  Heat  the  mixture  and 
stir  it  while  the  temperature  rises.  After  the  fat  has  been  melted, 
add  to  the  mixture  about  50  c.c.  of  potassium  hydroxid  solution 
(10  per  cent.)  and  boil  vigorously  until  saponification  is  complete, 
i.  e.,  until  the  solution  fulfills  the  conditions  mentioned  in  connec- 
tion with  process  126.     Typical  equations  : 

C15H31COO -CHj  CHjOH 

CisHjiCOO— CH    +  3K0H  =  3C15H31COOK  +   CIIOH 

I  I 

C15H31COO— CH,  CH.OH 

C,5H3iCOOH  +  KOH  =  CisHjiCOOK  +  H,0 

137-  Observe  the  frothiness  and  emulsifying  power  of  the  soap 
solution  just  produced  (127-128). 

*The  color  of  the  wax  is  due  to  a  pigment  and  not  to  the  fatty  matter  in  it. 
The  fragrant  odor,  also,  is  due  to  non-fatty  matter. 


38  Biochemical  Notes. 

138.  Liberation  of  the  fatty  acid.  To  the  hot  soapy  mixture 
produced  in  the  process  just  described  *  (136)  add  sufficient  concen- 
trated hydrochloric  acid  to  acidify  the  mixture  slightly.  Stir 
thoroughly  after  each  addition  of  mineral  acid  and  test  with  litmus 
strips  the  non-oleaginous  portion  of  the  mixture.  Notice  the  heavy 
layer  of  liquid  fatty  acid  that  is  finally  produced  (132).  After  re- 
moving the  stirring  rod  from  the  mixture,  pour  the  latter  into  a  small 
beaker  and  set  the  beaker  and  contents  in  a  water  bath  containing 
cold  water.  Cool  the  mixture  without  stilling  it,  so  as  to  favor 
solidification  of  the  palmitic  acid  in  the  form  of  a  thin  cake.  Keep 
Gold  water  in  the  bath. 

139.  Solution  of  the  fatty  acid.  After  the  palmitic  acid  has  be- 
come quite  solid  lift  the  cake  from  the  cold  subnatant  liquid.  Keep 
the  liquid  for  use  in  test  161.  Spray  the  cake  with  water  to 
wash  off  adherent  portions  of  the  slight  excess  of  hydrochloric  acid 
used  to  separate  the  palmitic  acid  from  the  soap,  break  the  cake  into 
small  pieces  and  transfer  the  pieces  to  about  250  c.c.  of  hot  95  per 
cent,  alcohol  in  a  beaker  on  a  water  bath.  Heat  the  alcoholic  mix- 
ture on  the  bath,  with  frequent  stirring,  until  most  of  the  palmitic 
acid  has  gone  into  solution,  when  the  alcohol  will  be  nearly  if  not 
wholly  saturated,  by  it  at  the  prevailing  temperature. 

140.  Crystallization  of  the  fatty  acid.  Immerse  in  hot  water 
(in  a  large  beaker)  a  small  beaker  whose  bottom  is  covered  with  a  thin 
layer  of  warm  95  per  cent,  alcohol.  In  the  latter  collect  the  pal- 
mitic acid  solution  after  its  passage  through  a  dry  filter  on  a  dry 
though  warm  funnel.  Do  not  pour  on  the  filter  any  oily  undis- 
solved palmitic  acid  in  the  alcoholic  mixture.  Let  the  alcoholic 
filtrate  cool  undisturbed.  Observe  the  cumulative  formation  of  a 
white  deposit  (palmitic  acid)  as  the  temperature  falls. 

141.  Purification  of  the  fatty  acid. — Examine  under  the  mi- 
croscope a  sample  of  the  palmitic  acid  deposited. f  Compare  with 
the  crystals  observed  in  experiments  lOO  and  loi.  Filter  the  mix- 
ture. Wash  the  crystals  free  from  adherent  acid  with  cold  95  per 
cent,  alcohol.     Notice  that  a  sample  of  the  alcoholic  filtrate  yields 

*  If  the  solution  was  permifcted  to  cool  perceptibly,  bring  it  again  to  the  boiling 
point  and  proceed  at  once  with  the  addition  of  hydrochloric  acid. 

t  Any  lauric  acid  (135)  that  may  be  present  in  the  myrtle  wax  tends  to  remain 
in  solution  at  this  point.     Lauric  acid  is  comparatively  soluble  in  cold  alcohol. 


Fats.  39 

a  precipitate  when  treated  with  water.  The  precipitate  consists  of 
palmitic  acid,  which  is  insoluble  in  water  (145).  The  remainder 
of  the  alcoholic  filtrate  will  be  collected  for  use  in  other  connections. 

142.  Redissolve  the  palmitic  acid  in  a  small  proportion  of  hot 
alcohol,  recrystallize  by  cooling  the  filtered  solution,  examine  under 
the  microscope  the  new  crop  of  crystals,  filter  oif  the  product  and 
wash  with  cold  95  per  cent,  alcohol  for  final  purification.  Dry  the 
washed  palmitic  acid  on  a  watch  glass  in  an  air  bath  at  about  40°  C, 
and  test  its  properties  by  the  methods  indicated  below. 

J.    Peoperties  of  a  Typical  Fatty  Acid  (Palmitic  Acid). 

143.  Reaction  to  litmus  (88). 

144.  Reaction  to  phenolphthalein.  —  Dissolve  some  of  the  pal- 
mitic acid  in  95  per  cent,  alcohol.     Warm  to  favor  solution. 

Into  5  c.c.  of  water  place  a  few  drops  of  phenolphthalein  solu- 
tion and  add  to  it  a  drop  or  two  of  very  dilute  sodium  carbonate 
solution  in  quantity  sufficient  to  induce  a  permanent  red  coloration. 

To  the  red  solution  add  the  palmitic  acid  solution  drop  by  drop 
until  acidity  is  indicated  by  the  discharge  of  the  color. 

145.  Solubilities  (65). 

146.  Melting  point  (no). 

147.  Acrolein  test  (113). 

148.  Conversion  into  soap.  (133)  Melt  the  remainder  of  the 
palmitic  acid  at  a  low  temperature  in  a  small  beaker.  Add  to  the 
oil  small  quantities  of  sodium  hydroxid  solution  with  repeated  stir- 
ring and  constant  warming,  until  the  mixture  is  made  slightly  alka- 
line in  reaction.  Pour  about  half  of  the  liquid  into  another  beaker 
and  let  it  cool.  A  hard  opaque  soap  is  obtained.  To  the  liquid 
portion  remaining  in  the  original  beaker  add  a  small  quantity  of 
alcohol,  stir  thoroughly  and  let  the  mixture  cool.  A  hard  trans- 
parent soap  is  obtained.  The  alcohol  exerts  merely  a  physical 
influence. 

149.  Dissolve  some  of  the  soap  in  water  and  apply  to  the  solu- 
tion tests  127  and  128. 

K.     Properties  of  Glycerol. 

Demonstrations.  150.  Tests  of  the  diffusibility  of  fats, 
fatty  acids,  soaps  and  glycerol.     151.  Preparation  of  glycerol 


40  Biochemical  Notes. 

on  a  large  scale,  and  identification  of  the  product.  152.  Solidi- 
fication of  glycerol.  151.  Distillation  of  glycerol  with  steam. 
154.  Borax  bead  test  for  glycerol. 

Test  small  quantities  of  commercial  glycerol  as  follows  : 

155.  Taste. 

156.  Reaction  to  litmus  (143). 

157.  Solubilities  (65). 

158.  Acrolein  test  (113). 

159.  Solvent  action  on  a  metallic  hydroxid.  Prepare  cupric 
bydroxid  by  adding  several  drops  of  potassium  hydroxid  solution  (10 
per  cent.)  to  about  10  c.c.  of  cupric  sulfate  solution  (2  per  cent.). 
Filter  and  wash  the  precipitate  with  water.  Mix  on  a  watch  glass 
a  drop  of  glycerol  with  a  drop  of  water.  On  a  second  watch  glass 
mix  a  drop  of  glycerol  with  a  drop  of  potassium  hydroxid  solution. 
Place  two  drops  of  water  on  a  third  watch  class.  To  the  liquid  on 
each  watch  glass  transfer  small,  approximately  equal  amounts  of  the 
washed  cupric  hydroxid.   Observe  the  solvent  action  of  the  glycerol.* 

160.  Dunstan's  test.  Add  to  5  c.c.  of  a  borax  solution  (5  per 
cent.)  a  quantity  of  alcoholic  solution  of  phenolphthalein  (1  per 
cent.)  sufficient  to  produce  a  permanent  and  distinct  red  color.  Add 
to  the  latter  drop  by  drop  aqueous  solution  (10  per  cent,  or  less)  of 
glycerol  until  the  red  color  is  discharged.  On  boiling  the  solution, 
the  red  color  returns.  Excess  of  glycerol  prevents  restoration  of 
the  color. 

At  present  this  reaction  cannot  be  satisfactorily  explained.  Am- 
monium salts  behave  like  glycerol  in  discharging  the  color,  but 
they  prevent  its  restoration.  The  reaction  is  given  by  polyhydric 
alcohols  in  general  (0:  84).  Among  the  latter,  sugars  are  conspicu- 
ous in  biological  materials.  Glycerol  may  be  separated  from  the 
sugars  by  the  distillation  method  already  demonstrated  (153). 

161.  Detection  of  glycerol  among  the  products  of  saponifi- 
cation. Filter  through  wet  paper  the  liquids  obtained  in  experi- 
ments 133  and  139.  Carefully  neutralize  both  filtrates  and  apply 
Dunstan's  test  to  each.     Demonstration  154  : 

*  There  is  probably  a  chemical  reaction  between  the  copper  cation  and  the  glyc- 
erol, with  the  formation  of  a  product  similar  to  an  alcoholate  (0  :  8i).  The  color 
is  doubtless  due  to  a  complex  copper-organic  ion  (L  :  226,  252). 

Make  a  larger  volume  of  the  copper-glycerol  solution  and  observe  that  boiling 
does  not  decompose  it  (L  :  226). 


Fats.  41 

L.       CaEBO HYDRATE    DERIVATIVES    OF    GLYCEROL. 

When  glycerol  is  carefully  oxidized,  e.  g.,  with  bromin  and  sodium 
hydroxid,  a  viscous  liquid  is  produced  that  shows  a  number  of  the 
properties  of  aldehydes  and  ketones.  The  product  is  called  "  glycer- 
ose  "  (crude)  and  contains  glyceric  aldehyde  and  dioxyacetone,  which 
are  isomeric  glyceroses. 

The  relations  of  these  products  to  glycerol  may  be  seen  at  a 
glance  below  : 

CH.,OH  CHO  CHjOH 

I     "  I  I 

CHOH  CHOH  CO 

I  I  I 

CH2OH  CHjOH  CH2OH 

Glycero  Glyceric  aldehyde  Di-oxyacetone 

Each  of  these  initial  oxidation  products  of  glycerol  has  many 
of  the  properties  of  the  simplest  sugars  and  each  is  a  carbohydrate. 

It  was  stated  above  that  crude  glycerose,  containing  glyceric  alde- 
hyde and  di-oxyacetone,  can  be  prepared  by  careful  oxidation  of 
glycerol  with  bromin  and  sodium  hydroxid.  The  latter  not  only 
assists  in  the  formation  of  the  oxidation  products  but  it  effects  a 
condensation  of  them  (0  :  106),  and  a  typical  sugar,  a-acrose,  re- 
sults.    Equation  : 


CHO  CHjOH  CHOH  CHOH 

I  II  I 

CHOH     +     CO  ^    CHOH         CO 

I  I  I  I 

CHjOK  CHjOH  CHjOH        CHjOH 

Glyceric       Di-oxyacetone  a-Acrose 

aldehyde  (i-Fructose) 

The  term  acrose  was  applied  to  this  sugar  because  the  product 
was  originally  obtained  from  acrolein  (113).  When  acrolein  is 
properly  treated  with  bromin,  two  hydroxyl  radicals  are  replaced 
by  bromin  atoms  and  di-bromacrolein  results  : 

CHO 

I  ^ 

CH^Br 

I 
CH,Br 

Di-bromacrolein 

Di-bromacrolein    yields   two  isomeric   sugars  on   treatment  with 


42  Biochemical  Notes. 

barium  hydroxide  one  of  which  is  a-acrose,  as  is  indicated  below  : 


2BaBr„ 


a-Acrose  is  a  constituent  of  crude  formose  (0  :  io8). 

These  observations  show  clearly  that  sugar  may  be  made  from  fat. 
Sugars  are  fermentable.  Among  the  products  of  fermentation  of 
sugars  are  glycerol  and  fatty  acids.  These  facts  show  that  fat  may 
he  produced  from  sugar. 

These  matters  will  be  discussed  in  subsequent  chapters  (second 
part)  and  in  the  lectures. 


CHO 

1 

i 
CHOH 

1 
CHOH 

1 

2  CH^Br    +     2Ba(OH)2    = 

=    CHOH 

1 
CO 

1 

CHsBr 

CHjOH 

CH^OH 

a-Acrose 

BIOCHEMICAL   NOTES: 


LABORATORY  WORK 


[Second  Part] 


BY 

WILLIAM  J.  GIES 


NEW  YOKK 
1906 


COPYKIGHT,  1906 

By  WILLIAM  J.  GIES 


Press  of 

The  new  Era  Printing  Companv 

Lancaster,  Pa, 


PREFACE. 

When  the  required  course  in  physiological  chemistry  at  the  Col- 
lege of  Physicians  and  Surgeons  was  started  early  in  February, 
1906,  the  first  part  only  of  this  volume  was  ready  for  immediate  use. 
The  second  part  of  the  volume  consists  of  two  additional  chapters 
that  could  be  conveniently  prepared  before  the  work  to  which  they 
relate  was  undertaken. 

William  J.  Gies. 
Laboratory  of  Physiological  Chemistey, 
College  of  Physicians  and  Surgeons, 
Columbia  Univehsity,  March  1,  1906. 


45 


CONTENTS. 

[Second  Paet.] 

Pagk 

CHAPTER  III.     Carbohydrates 47 

CHAPTER  IV.     Proteins 77 


46 


CHAPTER  III. 

CARBOHYDRATES. 

A.   Demonstration  of  Carbohydrate  Products. 

163.  Samples  of  typical  sugars,  dextrins,  glycogen,  gums, 
starch,  inulin  and  cellulose. 

B.    Chemical  Constitution  of  Carbohydrates. 

164.  General  elementary  composition   and   proportions. — 

Carbohydrates,  like  the  fats,  consist  of  carbon,  hydrogen  and 
oxygen.  The  fats  contain  relatively  small  proportions  of  oxygen 
and  comparatively  large  proportions  of  carbon  and  hydrogen.  The 
carbohydrates,  on  the  other  hand,  contain  relatively  small  propor- 
tions of  carbon  and  hydrogen  and  comparatively  large  proportions 
of  oxygen.  The  carbohydrates  represent  a  more  advanced  stage  of 
carbon-hydrogen  oxidation  (188)  than  the  fats,  and  therefore  are  as  a 
rule  less  prone  to  unite  with  additional  atoms  of  oxygen.  Conse- 
quently the  carbohydrates  possess  less  potential  energy  ;  they  yield 
less  heat  on  combustion.  The  subjoined  table  presents  results  that 
make  these  facts  quite  evident. 


Formula 

Percentage  Compo- 
sition 

Approximate 
Atomic  Ratios 

Heat  of 
Combus- 
tion :  cal- 

Typical carbohydrate : 

glucose.                    CgHijOg 

U          H          0 
40.00     6.66  53.34 

C:H      0:0      H: 
1  :2    1:1     2 

:0 
:1 

ories  per 
gram 

3763 

Typical  fat : 

tristearin.              C57Hi,oOg 

76.85  12.36  10.79 

1:2     9:1   18 

:1 

9530* 

The  conditions  indicated  above  account  for  the  well-known  in- 
flammability of  fats  (107)  and  the  fact  that  most  carbohydrates, 
sugars  especially,  do  not  take  fire  so  readily  (216).  Some  car- 
bohydrates, however,  such  as  cellulose,  which  contain  the  smallest 
proportions  of  oxygen  and  the  largest  proportions  of  carbon,  burn 
easily  and  without  sooty  flames.  The  very  large  proportions  of 
carbon  in  fats  account  for  the  usual  sootiness  of  the  flame  of  burn- 
ing fat  —  much  of  the  carbon  escapes  oxidation. 

*  Heat  of  combustion  of  mutton  tallow,  which  consists  largely  of  tristearin. 
That  of  pure  tristearin,  which  has  not  been  determined,  must  be  somewhat  higher. 

47 


48  Biochemical  Notes. 

Fats  and  polysaccharids  are  the  chief  carbonaceous  reserve  ma- 
terials in  organisms.  In  animals  both  classes  of  materials  are  par- 
ticularly involved  in  processes  connected  with  the  maintenance  of 
body  temperature. 

Practically  all  the  carbohydrates  contain  hydrogen  and  oxygen 
in  the  proportion  of  two  atoms  of  hydrogen  to  one  of  oxygen  — 
the  ratio  in  which  these  elements  occur  in  water.*  This  observa- 
tion led  promptly  to  the  selection  of  the  name  carbohydrate  (car- 
bon hydrate)  for  such  carbon  compounds. 

In  harmony  with  the  observation  just  referred  to  the  general 
composition  of  the  carbohydrates  may  be  expressed  by  the  formula 
C^(H20)  ,  in  which  x  and  y  represent  either  the  same  or  different 
multiples,  as  is  indicated  below  in  connection  with  the  formulas  of 
some  common  carbohydrates  (165)  : 

Glucose,  CgHijOg  =  Cfi(H20)g,  in  which  x  =  6  and  3/  =  6 
Sucrose,  CijHjjOn  =  Cjal  HjO)!,,  in  which  a;  =  12  and  2/  =  11 
Starch,  (C6Hio05),i=  [CgCHjOJs]™,  in  which  a;  =  n6  and  y—nb 

Such  formulas  mask  the  more  significant  intramolecular  relation- 
ships that  are  pointed  out  on  page  52. 

165.  Relative  elementary  composition  and  chemical  classifi- 
cation of  the  carbohydrates  of  greatest  biological  importance. 

A  general  classification  of  the  leading  carbohydrates  is  given  in  the 
table  on  page  49.     See  the  equations  in  section  169. 

166.  Relations  of  the  carbohydrates  to  polyhydric  alcohols. 
All  carbohydrates  may  be  regarded  as  oxy-derivatives  of  poly- 
hydric alcohols  (0  :  84). 

167.  Monosaccharids.  The  simplest  carbohydrates,  or  mono- 
saccharids  (165),  are  either  hydroxy-aldehydes  or  hydroxy- 
ketones  (0  :  97),  i.  e.,  aldehyde-alcohols  or  ketone-alcohols.  The 
monosaccharids  may  be  converted  by  reduction  into  their  correspond- 
ing polyhydric  alcohols,  just  as  certain  polyhydric  alcohols  may  be 
oxidized  to  their  corresponding  sugars  (169). 

All  monosaccharids  (and  all  other  carbohydrates)  contain  two  or 
more  hydroxyl  groups.     In  each  of  the  monosaccharids  one  of  the 

*  There  are  a  number  of  non-carbohydrate  substances,  such  as  acetic  acid, 
C2H4O2,  and  lactic  acid,  C3H6O3,  in  which  the  same  H  :  O  proportion  exists.  In  a 
few  unimportant  carbohydrates,  such  as  rhamnose,  C^H^fi^,  the  ratio  H  :  O  is  not 
the  same  as  in  water. 


a 

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Glycogen 

Dextrins 

Inulin 

13 

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02 

h-} 

<  y. 

ill  is 


OQ   O 


.2  -o 


t:      02      03 

«  5  S  So 


"^    '^    «« 


s  a 


"S    O    ic  -w"   ea 


0)     a 


c  ^  -^    c 


60 


Biochemical  Notes. 


hydroxyl  groups  is  directly  connected  with  a  carbon  atom  that  is 
linked  to  a  (a)  hydrogen  atom,  and  (6)  to  a  carbonyl  group  ;  and 
which  is  attached  by  its  fourth  bond  to  either  a  hydrogen  or  a  car- 
bon atom.  The  characteristic  group  in  every  monosaccharid  is  the 
following  : 

H— C— OH 

Characteristic  monosaccharid  group 

The  monosaccharids  of  the  aldehyde-alcohol  type  are  known  as 
aldoses.  Those  of  the  ketone-alcohol  type  are  called  ketoses.  In 
the  aldoses  the  carbonyl  group  is  linked  to  one  hydrogen  atom  and 
to  one  alcohol  group.  In  the  ketoses  the  carbonyl  group  is  held 
between  two  alcohol  groups. 

The  following:  constitutional  formulas  illustrate  these  relation- 
ships  : 


c— c— h| 

H 


Constitutional  formula  of  a  typical 
aldose 
Glucose 


Constitutional  formula  of  a  typical 

ketose 

Fructose 


The  constitutional  relations  between  typical  polyhydric  alcohols 
and  corresponding  monosaccharids  is  shown  below  : 


CH^OH 

(CH0H)3 

CHjOH 

Pentahydric  alcohol 
(C^H.^OJ 


CHO 

((^H0H)3 

CH2OH 

Pentose 
CaHioOs 


CHjjOH 

((JhOH), 

CH,OH 


CHO 


(i 


HOH), 


CH^OH 

Hexahydric  alcohol  Hexose :  aldose  Hexose :  ketose 

(CeHi^Oe)  (CeHi^Oe)  {CJi^fis) 


The  two  monosaccharid  groups  that  were  named  in  the  above 
summary,  i.  e.,  pentoses  and  hexoses,  are  the  most  important  bio- 
logically but  certain  additional  monosaccharid  groups  "^  are  of  general 
chemical  interest,  especially  in  connection  with  the  polyhydric  rela- 
tionships of  the  carbohydrates,  as  may  be  seen  on  the  opposite  page. 

*Each  of  these  special  groups  consists  of  laboratory  products.  None  of  them 
occurs  naturally.     Each  group  contains  aldose  and  ketose  representatives. 


Carbohydrates. 


51 


Alcohols : 

CH^OH 
CHOH 

CH^OH 

CH^OH 

CH2OH 

CH,OH 

CH,OH 
CHjOH 

((jjHOH), 

(CHOH), 

(([;HOH)e 

(inoH), 

CHjOH 

CH.OH 

CHjOH 

CH20H 

CHjOH 

Glycol 

Glycerol 

Erythrol 

Glucoheptol 

Glucoctol 

Glucononol 

Sugars : 

CHO 

CHO 
((^!HOH), 
CHjOH 

CHO 

CHO 
((jJHOH)^ 
CHjOH 

CHO 

CHO 

CHOH 

(CHOH), 

(CHOH), 
CH2OH 

CH2OH 

CHjOH 

CH2OH 

Glycolose 

Glycerose 

Erythrose 

Glucoheptose 

Glucoctose 

Glucononose 

Diose, 

Triose, 
C3H6O3 

Tetrose, 
C.HsO* 

Heptose, 

Octose, 

CsH.eOs 

Nonose, 
C.H„0, 

168.  Di-,  tri-,  tetra-  and  polysaccharids.  The  more  complex 
carbohydrates,  i.  e.,  those  not  included  in  the  raonosaccharid  group, 
are  anhydrids  of  aldehyde-alcohols  or  ketone-alcohols  or  both,  and 
may  be  readily  converted  by  hydration  into  hydroxy-aldehydes  or 
hydroxy-ketones  or  both,  i.  e.,  into  the  simplest  types  of  sugars, 
the  monosaccharids  (170).  The  carbohydrates  that  are  not  included 
in  the  monosaccharid  group  may  be  considered  as  anhydrids  of 
monosaccharids. 

169.  General  carbohydrate  relationships.  General  carbohy- 
drate relationships  are  indicated  by  the  following  equations  in 
which  effects  of  oxidation,  dehydration,  reduction  and  hydration  are 
shown  : 

Oxidation  (175) : 

CeHiA     +  O  =       CeHi.Oe       +     H,0 


Hexahydric 
alcohol 


^6'-'12'-'6 

Monosaccharid 
(Hexose) 


CsHijOs     +  O 

Pentahydric 
alcohol 


Dehydration  (165) 


Reduction : 


■'      C^HioOj       + 
Monosaccharid 
(Pentose) 


H,0 


SCfiHi^Oe-H^O 


C12H22O1] 
Disaccharid 


wCeHi^Os— "HjO  =    (CfiH.oO,)™ 

Polysaccharid 
(Hexosan) 


JiCsHioOa— JiHjO 


(C5HA).. 

Polysaccharid 

(Pentosan) 


CgHijOfi  +  H2  =  CgHitOg 

C5Hio05+H2=C,H,205 


52  Biochemical  Notes. 

Hydration  (165,  176) : 

(CeHioOs)^  +  nHjO  =.  nC^B.^O^ 

CnH,Ai  +H2O  -2C6H,A 
( CjHgO  J  „  +  «H,0  =  nCsHioOj 

170.  The  various  monosaceharid  derivatives  that  may  be  ob- 
tained by  simple  hydration  from  the  important  carbohydrates  of 
greater  complexity  are  indicated  in  the  following  summary  : 

Polysaccharids  :  Trisaccharid : 

Hexosans,  Raffinose  —  Glucose,  fructose 

Cellulose  *  —  Glucose  (n).  and  galactose. 

Starch  —  Glucose  {n).  Disaccharids  : 

Glycogen  —  Glucose  (w).  Sucrose  —  Glucose  and  fruc- 

Dextrin  *  —  Glucose  {n).  tose. 

Inulin  —  Fructose  (n).  Lactose  —  Glucose  and  galac- 

Pentosans,  tose. 

Araban  —  Arabinose  (w).  Maltose  —  Glucose  (2). 

Xylan  f  —  Xylose  (n).  Isomaltose  —  Glucose  (2). 

Constitutional  formulas  of  typical  carbohydrates.  171 .  Mono- 
saccharids.  The  appended  formulas  indicate  the  constitution  and 
the  stereochemical  relationships  of  some  of  the  monosaccharids  : 

CHO 

CHO       CHO       CHO     H— C— OH  H- 

H— C— OH  HO— C— H    H— C— OH  HO— C— H  HO- 

HO— C— H    H— C— OH  HO— C— H    H— C— OH  HO— ( 

HO— C— H    H— C— OH   H— C— OH   H— C— OH  H— C— OH 

CH,OH      CH,OH      CHjOH      CH^OH     CH2OH 

<-Arabinose  d-Arabinose  Z-Xylose  d-Glucose  d-Galactose 

The  members  of  each  main  group  of  carbohydrates  are  isomers. 
The  members  of  each  monosaceharid  subgroup,  such  as  the  aldohex- 
ose  group  (167),  have  the  same  constitutional  formula  and  are  stereo- 
isomers (0  :  28). 

172.  Disaccharids.  The  constitutional  formulas  of  some  disac- 
charids and  the  relations  of  disaccharids  to  monosaccharids  are 
shown  by  the  equations  on  the  opposite  page. 

*  Some  varieties  of  cellulose  and  dextrin  yield  mannose  (an  isomer  of  glucose) 
instead  of  glucose  ;  also  pentoses. 

tSome  varieties  of  xylan  yield  arabinose. 


Carbohydrates. 


63 


CHjOH 
CHOH 


CHOjH 
CHOH    :"*" 


CHOH 
HC==i0"" 


CHO 
CHOH 
CHOH 
CHOH 

CI 


CHO 
CHOH 


H,0 


HO— CH, 


^HOH 


Glucose 
(two  molecules) 


Maltose  * 
(one  molecule) 


H,0 


CHjOH 
CHOH 

CHO- 

--     I 
CHOH 

CHOH 

HC 


CHjOH 
CHO- 
CHOH 
CHOH 

CH,OH 


Glucose 
(one  molecule) 


Fructose 
(one  molecule) 


Sucrose  + 
(one  molecule) 


Each  of  the  above  formulas   for  disaccharids  may   be  written 
empirically  as  follows  : 

CeH„0-0-CeH„05 

173.   Trisaccharids.     The  constitutional  formula  of  raffinose  is 
indicated  below  : 


CHjOH 

i=C 


CHjOH 

(;hoh 


HC=0 


CHOH 

I  -2H,0: 

THOH 


CH^OH 


HC 


Glucose  Galactose  Fructose 

(one  molecule)    (one  molecule)    (one  molecule) 


RaflBnose 
(one  molecule) 


The  formula  for  raffinose  may  also  be  written  as  follows  : 
CeHiiOs-O-CsHioO^-O-CeHiiOs 

174.  Polysaccharids.    The  constitutional  formulas  of  the  polysac- 
charids  are  unknown.     That  they  are  analogous  to  those  of  the  di- 

*  The  formula  of  lactose  (composed  of  the  residues  of  a  molecule  of  glucose  and 
one  of  galactose)  is  essentially  the  same  as  that  of  maltose  (173)- 

t  Notice  the  fact  that  the  sucrose  formula  is  without  a  free  carbony  1  group  ( 225 ) . 


54 


Biochemical  Notes. 


and  trisaccharids  seems  probable.     The  empirical  formula  of  starch, 
(CgHjpOg)^,  may  be  written  as  follows  (173)  : 

C^HioOj-O CeHioO.-O-QHioO, O-C^HioOs 

Decomposition  products.  175.  Products  of  oxidation  (169). 
The  carbohydrates  may  be  readily  oxidized  to  carbon  dioxid  and 
water  but  all  of  them  yield  various  organic  acids  upon  less  vigorous 
oxidation.  Oxalic  acid,  for  example,  may  be  readily  produced  by 
profound  oxidation  (193).     Equation  : 

CHO 

CHOH 

I 

COOH 


CHOH 


i: 


+  90 


HOH 


CHOH 


A 


JH2OH 

Glucose 


3    I  +    3H,0 

COOH 


Oxalic 
acid 


Most  of  the  acids  that  result  from  direct  oxidation  of  the  car- 
bohydrates are  devoid  of  biological  interest.  The  following  formulas 
represent  the  most  important  types  of  acids  that  result  from  rel- 
atively slight  oxidation  : 


CHO 

CHO 
CHOH 

COOH 

1 

COOH 

CHOH 

CHOH 
CHOH 

CHOH 

CHOH 

CHOH 

CHOH 

CHOH 

CHOH 

CHOH 

CHOH 

1 

CHOH 
CH.OH 

CHOH 
COOH 

CHOH 
CH.OH 

CHOH 
COOH 

Glucose 

Glucuronic 
acid 

Gluconic 
acid 

Saccharic 
acid 

Of  the  three  acids  represented  by  the  above  formulas  glucuronic 
acid  is  particularly  important.     It  is  a  constituent  of  normal  urine. 

176.  Products  of  hydration  (169),  such  as  formic,  acetic,  pro- 
pionic, lactic,  butyric  acids  and  other  fatty  acids,  and  related  sub- 
stances, are  formed  from  carbohydrates  when  hydrolysis  is  carried 
to  the  point  of  decomposition  (0  :   135-140). 

177.  Glucosamin  is  an  important  carbohydrate  derivative.  It  is 
closely  related  both  to  hexoses  and  to  various  amino-acids  (0  :  184) 


Carbohydrates.  55 

that  may  be  derived  from  proteins  (250).     The  relation  between 
glucose  and  glucosamin  is  indicated  by  the  following  formulas : 

HC— OH 

CHjOH 

Glucose 

C.    Formation  of  Carbohydrates  (162). 

Laboratory  conversion  of  one  carbohydrate  into  another. 
178.  Polysaccharids.  Of  the  polysaccharids  named  in  the  table  on 
page  49,  only  dextrins  can  be  made  in  the  laboratory.  A  method 
for  the  preparation  of  dextrin  is  indicated  in  section  192. 

179.  Sugars.  Both  di-  and  monosaccharids  can  be  obtained 
from  polysaccharids  by  hydration  (169).  Monosaccharids  can  be 
obtained  from  the  more  complex  sugars  by  the  same  process  (169). 

Maltose  has  been  made  from  concentrated  solutions  of  glucose  by 
hydration  through  the  agency  of  an  enzyme  called  maltase.  This 
enzyme  is  also  able  to  bring  about  the  conversion  of  maltose  into 
glucose.  The  hydration  process  that  is  induced  by  maltase  is 
obviously  reversible  and  may  be  represented  thus  : 


C,,H„0„  +  H,0  ^  2C6H,  A 

Maltose  Glucose 

Laboratory  production  of  carbohydrates  from  non-carbohy- 
drates. 180.  The  monosaccharids  have  been  made  in  the  labora- 
tory from  non-carbohydrate  materials  by  several  methods.  With 
the  exception  of  maltose  (179)  none  of  the  higher  carbohydrates 
named  on  page  49  has  been  produced  synthetically.  The  transfor- 
mation of  polyhydric  alcohols  by  oxidation  into  sugars  has  already 
been  referred  to  (169). 

181.  Synthesis  of  i-Fructose.  At  the  close  of  the  preceding 
chapter  (Fats,  page  41)  attention  was  drawn  to  some  carbohydrate 
derivatives  of  glycerol.  Allusion  was  made  to  facts  showing  that 
carbohydrate  may  be  made  from  fat. 

The  formulas  and  equations  on  page  41  (first  part  of  the  volume) 
explain  the  derivation  of  glyceroses  (trioses),  from  glycerol  and  also 
the  condensation  of  i-fructose  (a-acrose),  from  the  glyceroses :  glyceric 


56  Biochemical  Notes. 

aldehyde  and  di-oxyacetone.  It  was  stated,  in  connection  with  the 
equation  showing  this  condensation,  that  ^-fructose  is  a  constituent 
of  crude  forraose  (0  :  lo8). 

182.  Syniheds  of  crude  formose.  Treated  with  saturated  lime 
water  for  several  days  at  room  temperature,  formaldehyde  gradually 
undergoes  polymerization  (by  aldol  condensation,  0  :  106).  The 
product  of  the  polymerization  is  a  sweet  syrup,  which  is  called 
crude  formose,  and  contains  formoses  and  a-acrose.     Equation  : 

6H— CHO    =    CgHiPe 

The  condensation  may  be  represented  graphically  as  follows  : 

H  H 


H— C=0 

H- 

-C— OH 

H— CHO 

CHOH 

H— CHO 

= 

CHOH 

H— CHO 
H— CHO 

CHOH 

1 
CHOH 

1 

H— CHO 

CHO 

Formaldehyde 
(six  molecules) 

Formose 
(one  molecule) 

183.  Natural  formation  of  carbohydrates  from  non-carbo- 
hydrates. The  synthesis  of  hexoses  from  formaldehyde  (182)  has 
an  important  bearing  on  the  production  of  carbohydrates  from 
inorganic  matter  in  plants. 

Carbon  dioxid  is  absorbed  from  the  air  into  plants.  Water  is 
present  in  large  proportions  throughout  all  the  green  parts  of  living 
plants.  The  chlorophyl  (green  coloring  matter)  of  plants  has  the 
power,  under  the  influence  of  sunlight,  to  effect  combinations 
between  carbon  dioxid  ah3  water.  It  is  generally  believed  that 
formaldehyde  is  produced  by  reduction  from  the  carbon  dioxid  and 
water,  that  the  resultant  formaldehyde  is  polymerized  into  glucose 
and  that  the  latter  is  dehydrated  into  more  complex  carbohydrates, 
such  as  starch.  These  chemical  changes  doubtless  occur  in  har- 
mony with  the  following  equations  : 

CO2  +  H,0  =  H— CHO  +  Oj 
6H— CHO  =  CgHiA  [182] 
nCeHiA— wH^O  =l^^Y{^^0^)n  [169] 


Carbohydrates.  57 

The  probability  that  these  deductions  are  correct  is  increased 
by  the  fact  that  traces  of  formaldehyde  have  been  detected  in  the 
green  parts  of  plants.  It  has  been  shown  that  in  certain  sea  weeds 
(Spirogyra),  formaldehyde  sodium  bi-sulfite  (0 :  lo8)  is  decomposed 
by  the  living  cells,  and  that  the  liberated  formaldehyde  is  imme- 
diately condensed  to  sugar  and  precipitated  in  the  cells  as  starch.* 

184.  Glucosids.  Acetals.  Aldehydes  unite  with  alcohols,  in  the 
proportion  of  one  molecule  of  the  former  to  two  of  the  latter,  with 
elimination  of  water,  to  form  acetals  (0  :  105).  The  reaction  is 
induced  by  mineral  acid.     Equation  : 


i        ii:u— Uon-  /^ — CjHi 

CH3— CH:0+     i         '    "  =  CH,— CH<  +  H,0 

i HiO— CjHj  \0— CjHj 

Acetaldehyde     Ethyl  alcohol  Acetal 

(two  molecules) 

185.  Synthdic glucosids.  Glucose  can  be  brought  into  union  with 
alcohols,  in  a  reaction  similar  to  that  shown  above  (184),  to  form  a 
group  of  substances  called  glucosids,  which  are  analogous  to  the 
natural  glucosids.  f     Typical  equations  : 


H    HjHiH    H, H  H  H 

n  Yj  v-v J     ?j  n 

a.  H— C— C— C— (J— C— C   +  I  H;0— C— H  =  H— 0— C— C— C- 


CgHiA  CH3OH  CeHnO^-O— CH, 

Glucose  Methyl  alcohol  Methyl  glucosid 

6.  CeHiA  +  CsH^COH),  =  CeHnOs-O-C^HjCOH),   +  H,0 

Glycerol  Glycero-glucosid 

In  these  reactions  only  one  alcohol  molecule  unites  with  a  single 
aldose  molecule.    One  of  the  hydroxyl  groups  of  the  latter  plays 

*  These  facts  suggest  that  formaldehyde,  if  produced  in  plants,  as  seems  to  be 
indicated,  is  a  very  transient  product  and  under  normal  conditions  never  accumu- 
lates in  suflScient  proportion  to  exert  its  well-known  destructive  effects  on  proto- 
plasm. 

fThe  natural  glucosids  are  carbohydrate  esters,  i.  e.,  "combined"  carbohy- 
drates. On  hydration  with  acids  or  enzymes,  or  by  electrolytic  cleavage,  mono- 
eaccharids  are  produced  from  them.  As  a  rule  the  term  glucosid  is  restricted  to 
plant  products  of  this  nature,  of  which  there  are  many  varieties.  In  a  general 
way  the  term  is  sometimes  applied  to  all  substances  that  are  not  true  carbohy- 


58  Biochemical,  Notes. 

the  part  usually  taken  by  the  second  molecule  of  alcohol  in  acetal 
formations.  On  hydration  of  the  glucosid  the  aldose  and  alcohol 
are  regenerated  ;  thus  (0  :  i6o)  : 

CfiHnOs-O-CHj  +  H^O  =  CeHiPe  +  CH3OH 

The  combination  of  two  monosaccharid  molecules  with  elimina- 
tion of  a  molecule  of  water  to  form  a  disaccharid  (172)  is  analogous 
to  the  production  of  a  glucosid  from  an  alcohol  and  a  monosaccharid. 

D.   Relationship  Between  Fat  and  Carbohydrate. 

186.  Fats  may  be  converted  into  carbohydrates  and  vice 
versa.  Both  fats  and  carbohydrates  yield  glycerol  and  fatty 
acids  under  favorable  conditions  of  decomposition.  At  the  conclu- 
sion of  the  preceding  chapter,  on  fats,  it  was  stated  that  carbo- 
hydrates may  be  made  from  fats.  The  production  of  glyceroses 
and  a-acrose  from  glycerol  was  explained.  We  have  seen  how  the 
simplest  fatty  acid  may  be  converted  into  carbohydrate  (182).  In 
our  study  of  metabolism  we  shall  learn  that  other  fatty  acids  than 
formic  acid  appear  to  be  convertible  into  carbohydrate.  Synthesis  of 
fats  from  fatty  acids  and  glycerol  may  be  effected  without  difficulty. 

187.  Relative  degrees  of  oxidation.  Fat  may  arise  in  organ- 
isms from  carbohydrates  by  processes  of  reduction  or  dehydration^ 
and  carbohydrates  may  be  formed  in  organisms  from  fat  by  proc- 
esses of  oxidation  or  hydration.  The  general  chemical  relationship 
existing  between  fats  and  carbohydrates  may  be  understood  from  a 
study  of  typical  formulas. 

An  examination  of  the  formulas  of  typical  fats  and  carbohy- 
drates shows  at  a  glance  the  great  difference  in  the  degree  of  oxida- 
tion of  the  two  classes  of  substances  (164)  : 

drates,  but  which,  on  decomposition,  yield  monosaccharid  molecules.  In  the 
latter  sense  even  some  proteins,  like  mucoids  (250),  are  glucoaids. 

Typical  natural  glucosids  and  their  initial  cleavage  products  are  named  below  : 

CisHigO,  +  HjO  =  CjHgOj  +  CgHijOe 
Salicin  Saligenin       Glucose 

CjoHj7NO,i  +  2H2O  =■  CfiHs— CHO  +  HON  +  2C6Hi,06 

Amygdalin  Benzal-  Hydrocy- 

dehyde  anic  acid 

CioHjeNS.OgK  +  H,0  =  C3H5-NCS  +  KHSO,  +  C^B.^fi^ 

Sinigrin  Allyl  mus- 

( Potassium  myronate)  tard  oil 


Carbohydrates.  59 

Tristearin,  C57H,iq06       Percentage  of  oxygen  is  10.79. 
Glucose,       CgHjjOg  Percentage  of  oxygen  is  53.33. 

1 88.  Transformation  of  sugar  into  fat.  Since  fat  is  formed 
in  organisms  from  carbohydrate  it  may  be  presumed  that  glucose 
or  glucose-yielding  material  furnishes  the  starting  point  for  such  a 
production.  It  is  possible  also  that  glucose  fragments  are  utilized 
in  the  process.  In  plants  the  starting  point  may  be  formaldehyde 
synthesized  from  carbon  dioxid  and  water  (182). 

If  we  assume,  for  purposes  of  illustration,  that  glucose  can  be 
converted  into  any  or  all  of  the  common  fats,  it  is  desirable  to 
ascertain  the  simplest  terms  in  which  such  a  transformation  of  glu- 
cose may  be  expressed. 

Assuming,  further,  that  all  of  the  carbon  of  the  glucose  is  util- 
ized in  such  a  production  of  fat,  it  is  obvious  that  at  least  9J  mole- 
cules of  glucose  would  be  necessary  for  the  synthesis  of  tristearin. 
The  following  calculations  give  the  simplest  terms  in  which  this 
process  may  be  conceived  to  occur : 

Formnla  of  tristearin Cj^HjioOj 

Corresponding  number  of  atoms  composing  9^  mole- 
cules of  glucose C57HU4O57 

Excess  of  atoms  over  the  immediate  synthetic  require- 
ments   H4O51)  or,  2HjO  +  49  0 

These  calculations  imply  that  a  formation  of  fat  from  glucose 
must  be  attended  by  very  decided  reduction  of  the  carbohydrate. 

The  above  calculations  and  similar  data  for  such  syntheses  of 
other  fats  may  be  expressed  empirically  as  follows : 

Tristearin,  9^C6H,  A  =  C5,Hiio06  +  2H,0  +  49  0 

Triolein,  g^CgHijOe  =  C57H10A  4-  5HjO  +  45  0 

Tripalmitin,  SJCgHiA  =  CsiHjgOe  +  2HjO  +  43  0 

Tributyrin,  2^C6Hi  A  =  C.^H^fi^  +  2H2O  +70 

The  above  facts  may  also  be  expressed  empirically  in  a  manner 
illustrated  by  the  following  equation  : 

igCfiHijOe  -t-  98H2  =  Cs^HiioOe  +  IO2H2O 

The  striking  facts  in  the  above  quartette  of  equations  are  (a)  the 
large  residues  of  oxygen,  which  emphasize  the  great  degree  of  reduc- 
tion necessary  to  convert  sugar  into  fat,  and  (6)  the  half-molecular 


60 


Biochemical  Notes. 


quantity  of  glucose-carbon  necessary  to  make  up  the  full  quota  of 
fat-carbon .  The  constancy  of  the  half-molecular  quantity  of  glucose- 
carbon  required  by  the  simplest  terms  of  the  synthesis,  suggests  that 
this  carbon  residue  is  utilized  for  the  production  of  the  glycerol  part 
of  the  fat  molecule,  a  deduction  that  is  emphasized  by  the  water  mole- 


o 

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w 

Carbohydrates.  61 

cules  of  each  equation,  which  seeraiDgly  result  from  a  glycerol-fatty 
acid  combination   such  as  occurs  in  ester  formations   in   general 

(0:  154). 

These  deductions  seem  to  be  verified  by  the  graphic  arrange- 
ments and  empirical  suggestions  on  the  preceding  page. 

E.   Typical  Carbohydrates  for  Use   in  the  Tests. 

189.  Kinds.  In  experiments  206-249  use  solid  samples  or 
2  per  cent,  solutions  of  each  of  the  following  typical  carbohydrates  : 
Glucose,  sucrose  and  dextrin.  Use  also  solid  starch  or  2  per  cent, 
starch  paste  (194).  *  Practically  all  the  properties  of  the  various 
important  kinds  of  carbohydrates  that  are  of  biochemical  interest 
are  illustrated  by  these  four  typical  products. 

Preparation.  190.  Glucose.  The  typical  monosaccharid  occurs, 
like  nearly  all  the  carbohydrates,  widely  distributed  in  plants. 
Nevertheless  it  cannot  be  obtained  very  conveniently  in  large  quan- 
tities from  plants  and  is  commonly  manufactured  on  a  large  scale 
from  starch  or  sucrose  by  a  general  process  similar  to  the  following  : 
Starch  is  boiled  with  comparatively  dilute  sulfuric  acid.  In  this 
process  starch  is  hydrated  into  various  simpler  carbohydrates,  in- 
cluding glucose  (170).  The  thoroughly  hydrated  mixture  is 
neutralized  with  calcium  carbonate  and  filtered  through  animal 
charcoal  to  remove  calcium  sulfate  and  also  colored  matters  that 
were  developed  as  by-products  in  the  heating  process.  The  de- 
colorized solution  is  evaporated  to  the  proper  consistency  and  on 
cooling  crude  glucose  solidifies  as  an  amorphous  mass.  The  crude 
product  always  contains  dextrins,  which  may  be  removed  by  treat- 
ing the  mass  with  hot  90  per  cent,  alcohol.  Dextrins  do  not  dis- 
solve in  the  alcoholic  liquid,  glucose  does.  Glucose  may  be  ob- 
tained by  evaporation  of  the  alcoholic  filtrate. 

191.  Sucrose  is  usually  obtained  from  the  sugar-cane  or  beet- 
root. The  former  contains  about  16-18  per  cent,  of  sucrose,  the 
latter  about  13-14  per  cent.  A  general  process  that  is  employed  for 
the  preparation  of  sucrose  is  the  following  :  The  juice  or  hot  aqueous 
extracts  of  the  cane  or  root  are  obtained.  The  liquid  is  boiled  with 
milk  of  lime,  in  which  process  proteins  are  coagulated  (322)  and 

*  Other  important  carbohydrates,  such  as  glycogen  and  maltose,  will  receive 
due  attention  later. 


62  Biochemical  Notes. 

lime  salts  of  various  acids  such  as  oxalic  and  phosphoric  are  pre- 
cipitated. The  filtrate  is  treated  with  carbon  dioxid  to  precipitate 
calcium  ;  *  some  decolorization  also  results.  The  filtrate  from  the 
calcium  precipitate  is  then  evaporated  to  the  proper  consistency  for 
crystallization  of  the  sugar.  Ordinary  molasses  is  the  syrupy 
mother  liquor  drained  from  the  crystals  of  crude  sucrose  obtained 
in  this  manner. 

Crude  sucrose  has  a  brown  color.  In  the  refining  process  the 
aqueous  solution  of  the  crude  product  is  heated  with  milk  of  lime 
or  some  other  decolorizing  agent.  The  clarified  liquid  is  filtered 
through  animal  charcoal  and  the  pure  sugar  crystallized  from  the 
colorless  filtrate. 

192.  Dextrin.  Commercial  dextrin,  consisting  chiefly  of  a 
mixture  of  several  dextrins,  is  usually  prepared  by  a  process  simi- 
lar to  the  one  described  above  for  the  production  of  glucose  (190). 
During  the  heating  process  samples  of  the  acid  mixture  (starch  and 
dilute  sulfuric  acid)  ar^  repeatedly  treated  with  iodin  (196).  As  a 
rule  the  hydration  process  is  stopped  as  soon  as  a  sample  of  the 
mixture  fails  to  yield  a  blue  color  with  iodin.  At  this  point  the 
dextrin  is  precipitated  with  alcohol  (196,  239).  This  precipitate 
frequently  contains  a  little  glucose,  and  also  soluble  starch  if  the 
hydration  process  was  not  carried  far  enough.  Glucose  may  be 
removed  by  repeated  reprecipitation  of  the  dextrin  (from  aqueous 
solution)  with  alcohol  and  the  soluble  starch  can  be  eliminated  by 
further  treatment  of  the  product  with  hot  acid. 

Demonstrations.  193.  Preparation  of  oxalic  acid  (175)  from 
carbohydrate.  194.  Preparation  of  starch  from  potato  and 
of  ' '  starch  paste  "  from  the  starch  thus  obtained.  195.  Micro- 
scopic characters  of  various  forms  of  starch.  196.  Preparation 
of  glucose  and  dextrins  from  starch  by  methods  190  and  192, 
with  observations  of  the  effects  of  iodin  and  of  alcohol  on  samples 
of  the  mixtures  taken  at  brief  intervals.  197.  Preparation  of 
glucose  and  fructose  from  sucrose  and  determination  of  the 
effect  on  sucrose  of  boiling  concentrated  hydrochloric  acid. 


*  Soluble  carbohydrates  unite  with  calcium  hydroxid  and  other  alkalies  to 
form  compounds  similar  to  alcoholates  (0:8i).  They  are  called  glucosates, 
Bucrates  and  so  on.     Each  is  decomposed  by  carbon  dioxid.     Typical  formulas  : 

CgHiA,  CaO  C12H2JO11.  2CaO 

Calcium  glucosate  Calcium  sucrate 


Carbohydrates.  63 

198.  The  polariscope  and  optical  properties  of  carbohydrates. 

199.  Tests  of  the  diffusibility  of  carbohydrates.  200.  Tests 
of  the  fermentability  of  carbohydrates,  with  qualitative  and 
quantitative    determinations    of   products    of    fermentation. 

201.  Effects  upon  carbohydrates  of  different  kinds  of  bacteria. 

202.  Properties  of  cellulose.  203.  Preparation  of  pentosan 
and  pentose.  204.  Production  of  furol  from  pentoses  and 
pentosans,  and  application  of  the  anilin  test  for  furol.  205. 
Furol  and  its  properties. 

F.    Elementary  Composition  of  Carbohydrates. 

206.  Carbohydrates  consist  wholly  of  carbon,  hydrogen  and 
oxygen.  Apply  tests  50  and  51  to  a  sample  of  one  of  the  products. 
Prove  the  absence  of  nitrogen,  sulfur,  phosphorus  and  iron,  tests 
53,  62,  63,  64.     Compare  with  86. 

G.    Physical  Properties  of  Carbohydrates. 

207.  Carbohydrates,  like  fats,  are  contained  in  all  organisms. 
Fats  are  more  conspicuous  in  animals,  carbohydrates  in  plants. 

Fats  are  very  much  alike  in  physical  and  chemical  properties. 
Carbohydrates,  on  the  other  hand,  differ  considerably,  both  physi- 
cally and  chemically. 

208.  Carbohydrates  do  not  impart  greasy  stains  to  paper. 
(87).* 

209.  Pure  carbohydrates  are  neutral  compounds  (88). 

210.  Microscopic  appearance.  Examine  under  the  microscope 
powdered  samples  of  the  carbohydrates  (215). 

211.  Solubilities  (65). 

212.  Carbohydrates  do  not  form  emulsions  (93). 

213.  Examine  starch  paste  under  the  microscope  (195). 
Crystallinity.     214.  Demonstration.     Methods  for  crystal- 
lizing carbohydrates. 

215.  Examine  under  the  microscope  a  sample  of  one  of  the 
crystallized  sugars. 

Demonstrations.  216.  Effects  of  heat  on  carbohydrates 
and  an  examination  of  the  products  of  their  destructive  distil- 
lation.    217.  Determination  of  the  melting  point  of  a  sugar. 

*  Eepeat  the  test  indicated  and  notice  agreement  or  disagreement  with  former 
results. 


64  Biochemical  Notes. 

H.    Chemical  Properties  or  Carbohydrates. 

Demonstrations.  218.  Microscopic  appearance  of  variqus 
solid  carbohydrates  treated  witli  iodin.  219.  The  phloroglucin 
and  orcin  coloration  tests  for  pentoses  and  pentose-yielding  sub- 
stances. 220.  The  resorcin  coloration  test  for  fructose  and 
fructose-yielding  substances.  221.  Precipitation  of  carbo- 
hydrates from  alkaline  solutions  with  benzoyl  chlorid  and 
methods  for  the  recovery  of  the  carbohydrates  from  the  benzoic 
acid  esters  thus  produced. 

Coloration  tests.  222.  3Iolisch-v.  Udrdnszky  test.  To  about  2 
c.c.  of  the  carbohydrate  solution  in  a  test-tube  add  2  drops  of  a  10 
per  cent,  alcoholic  solution  of  a-naphthol.  Shake  the  mixture. 
Pour  carefully  and  gradually  down  the  side  of  the  tube  about 
2-4  c.c.  of  concentrated  sulfuric  acid.  At  the  line  of  junction  of 
the  two  liquids  a  violet  or  raspberry-red  coloration  will  appear  if 
the  test  is  positive.     Shake  the  mixture.     Add  more  acid. 

This  is  a  color  test  {or  Jurol.*  All  carbohydrates  yield  furol  on 
treatment  with  concentrated  sulfuric  acid.  Many  proteins  yield  furol 
under  the  same  treatment  (306).  The  test  is  given  not  only  by  furol 
but  also  by  all  carbohydrates,  and  all  the  furol-yielding  substances. 

The  characteristic  colorations  are  due  to  compounds  of  a-naphthol 
and  furol,  the  exact  natures  of  which  have  not  been  ascertained. 
The  chemical  characters  of  furol  and  a-naphthol  are  indicated  by 
the  following  formulas  : 

H        H 
C         C 

HC^^^^^'^^CH  /CH=CH  HC— CH 

HC  CH       "     "^^""^^g— Ah  J      fi-CHO 

c       c  "" 

H        OH 

a-Naphthol  Furol 

CioH,OH  C^HjO— CHO 

The  formation  of  furol  from  a  typical  carbohydrate  (pentose),  by 
the  action  of  mineral  acids  such  as  sulfuric  and  hydrochloric 
(demonstration  204),  is  indicated  by  the  equation  on  the  opposite 
page. 

*Also  called  furfurol,  furfuraldehyde  and  furfural. 


Caebohydrates. 


OH 

HC— 


H 
-CH 

OH 
OH 


HC— 

I 
OH 


!— CHO 


H 


;  OH 

I- 


"?- 


H  i 

!• 

OjH 
OH 


HC=CH 


—  3H,0  = 


CHO 


Pentose  (171) 
(Formula  curled  up) 


OH  H 

Pentose 


HC=0— CHO 


Furol 


223.  lodm  test.  A  few  polysaccharids  yield,  with  iodin,  blue  or 
red  colored  products,  which  contain  iodin  in  variable  proportions  and 
combinations.  None  of  the  sugars  forms  colored  products  with 
iodin. 

Add  to  the  solids  and  to  the  liquids  (189)  ordinary  "  iodin  solu- 
tion," i.  e.,  iodin  dissolved  in  potassium  iodid  solution.*  Divide 
into  four  parts  the  colored  solutions  containing  iodids  of  polysac- 
charids and  treat  the  parts  as  follows  : 

a.  Heat  one  portion  to  boiling.  Cool.  Add  an  equal  volume 
of  fairly  concentrated  albumin  solution.     Heat  again,  then  cool. 

b.  Make  a  second  portion  slightly  alkaline.  Acidify.  Render 
alkaline. 

c.  Treat  a  third  portion  with  a  small  amount  of  alcohol.  Dilute 
greatly  with  water. 

d.  To  the  remaining  portion  add  solution  of  silver  nitrate  in 
moderate  excess. 

224.  Moore^s  test.  Boil  with  an  equal  volume  of  potassium 
hydroxid  solution.  A  yellow  to  deep  brown  coloration  may  re- 
sult. If  heated  long  enough  a  resinous  mass  may  be  deposited  as 
in  the  case  of  aldehydes  (0 :   106). 

Coloration  in  Moore's  test  is  due  to  oxidation  of  the  carbohy- 
drate with  formation  of  alkali  salts  of  glucic  acid,  C,2Hjg09  and  sac- 
charumic  acid,  Cj^HjgO,j  ('?),  each  of  which  has  a  brownish  color. 

This  reaction  is  given  only  by  the  carbohydrates  that  contain  free 
carbonyl  groups  (172).  Consequently  sucrose  and  the  polysac- 
charids do  not  yield  it.  The  carbohydrates  that  yield  the  reaction 
are  quickly  destroyed  by  the  reagent. 

*  The  reaction  given  by  starch  cannot  be  obtained  with  a  pure  aqueous  solu- 
tion of  iodiu. 


66  Biochemical  Notes. 

Reduction  tests.  225.  All  the  carbohydrates  that  contain  free 
carbonyl  groups  exhibit  the  property  of  reducing  certain  compounds 
of  heavy  metals,  especially  when  the  latter  are  dissolved  in  alkaline 
liquids  (172). 

The  reduction  tests  are  seriously  impaired  by  various  substances, 
among  the  most  common  of  which  are  acids,  ammonium  salts  and 
proteins  (288). 

Acid  solutions  that  are  to  be  tested  must  always  be  made  neutral 
or  slightly  alkaline  (preferably  with  an  alkali  hydroxid,  never  with 
ammonium  hydroxid),  before  adding  the  alkaline  metallic  solution, 
because  acids  themselves  exert  reducing  action  on  the  compounds  of 
the  heavy  metals  employed  in  the  tests  and  also  diminish,  in  some 
cases  may  completely  neutralize,  the  required  alkalinity  of  the  solu- 
tion of  the  metallic  compound. 

Ammonium  compounds  are  acted  upon  by  the  hydroxid  in  the 
solution  of  the  heavy  metal,  with  consequent  production  of  free 
ammonia.  Before  using  the  alkaline  solution  of  the  metallic  com- 
pound sufficient  alkali  hydroxid  must  be  added  to  the  solution 
under  examination  to  effect  complete  decomposition  of  any  ammo- 
nium compounds  present  in  it  and  to  insure  the  presence  of  a  mod- 
erate excess  of  the  reagent. 

Proteins  tend  to  hold  the  metallic  reduction  products  in  solution 
(298),  or  yield  precipitates  in  the  alkaline  liquids  that  resemble  the 
reduction  products  (233).  As  a  rule  it  is  necessary  to  make  cer- 
tain that  proteins  are  absent  (297)  before  applying  the  reduction 
tests  described  below. 

226.  Silver  mirror  test.  Clean  a  test-tube  thoroughly  by  boiling 
in  it  concentrated  nitric  acid,  afterward  potassium  hydroxid. 

To  about  5  c.c.  of  silver  nitrate  solution  in  the  perfectly  clean 
tube  add  a  few  drops  of  ammonium  hydroxid,  i.  e.,  just  sufficient  to 
dissolve  the  precipitated  silver  oxid.     Equations  : 

2AgN03     +     2NH^0H    =    Ag.O    +    2NHiN03     +     H^O 
Silver  oxid 

Agfi     +     4NH4OH     =    2Ag(NH3)20H     +     3H2O 
Silver-ammonium 
hydroxid 

To  the  alkaline  solution  of  silver-ammonium  hydroxid  thus  pre- 
pared add  a  few  drops  of  the  carbohydrate  solution.  Heat  the  tube 
and  contents  in  boiling  water.     If  reduction  occurs  it  is  shown  at 


Carbohydrates.  67 

once  or  after  a  few  minutes  by  deposition  of  metallic  silver,  usually 
in  the  form  of  a  mirror,  if  the  tube  was  perfectly  clean  at  the  be- 
ginning of  the  experiment.     Typical  equation  (L  :  225  )  : 


* 


2Ag(NH3),OH  +  3H,0  +  R— CHO  =  2Ag  +  R-COONH,  +  3NH,0H  +  H^O 

227.  Trovnner's  test.  To  about  3  c.c.  of  potassium  hydroxid 
solution  (10  per  cent.)  add  several  drops  of  a  solution  of  cupric 
sulfate  (1  per  cent.).     Equation  : 

CuSO^      +     2K0H      =     Cu(OH),      +      KjSO^ 
Cupric  hydroxid 

To  the  slightly  bluish  mixture  just  prepared  add  about  an  equal 
volume  of  carbohydrate  solution.  Sugars  manifest  solvent  action 
on  the  cupric  precipitate  (159). 

Boil  the  liquid  for  a  minute  or  two.  If  reduction  occurs  the  blue 
color  disappears  or  is  diminished  in  intensity,  and  a  yellow  or,  more 
frequently,  a  red  precipitate  is  produced.     Equations  : 

/0H,_> 

^Ni  o^  w:  Cu— OH 

\^::i^:  -0=1  +  H,o 

Cu/i^^ i  ^^-^° 

\0H 

Cupric  hydroxid  Cuprous 

(two  molecules)  hydroxid 

[Soluble  :  blue]  [Insoluble  :  yellow] 

Cu— OH  Cu. 

I  —   HjO    =       i    >0 

Cu— OH  Cu/ 

Cuprous  oxid 
[Insoluble:  red] 

It  frequently  happens  that  the  boiled  mixture  exhibits  other 
colors,  such  as  green  and  brown,  which  are  due  to  mixtures  of  some 
of  the  colors  already  referred  to.  On  allowing  the  mixture  to  stand, 
the  reduction  product  quickly  subsides. 

Tlie  copjier  compound  is  reduced  by  the  carbohydrate.  The  carbo- 
hydrate is  oxidized  by  the  copper  compound,  with  effects  on  the  car- 
bohydrate similar  to  those  in  Moore's  test  (224). 

When  only  very  slight  proportions  of  cupric  hydroxid  and  re- 
ducing carbohydrate  are  present  together  in  a  mixture,  boiling  may 
result  in  complete  discharge  of  the  faint  blue  color  or  in  entire  re- 
placement of  it  by  a  yellowish  tint,  without  causing  a  cuprous 

*R  is  intended  to  represent  the  main  part  of  the  carbohydrate. 


68  Biochemical  Notes. 

deposit.  In  such  cases  the  cuprous  compound  is  held  in  solution. 
The  yellowish  tint  results  from  the  action  of  the  alkali  hydroxid  on 
the  carbohydrate,  as  in  Moore's  test  (224). 

Precautions.  Care  must  be  taken  to  prevent  the  presence  of  ex- 
cess of  cupric  hydroxid.  The  deeper  the  blue  color  of  the  mixture 
the  greater  the  difficulty  of  detecting  a  cuprous  precipitate,  partic- 
ularly when  only  a  slight  proportion  of  reducing  carbohydrate  is 
present  and  the  amount  of  resultant  precipitate  is  relatively  slight. 
Excess  of  copper  may  be  deposited,  on  boiling,  as  a  hlach  precipitate 
of  cupric  oxid,  which  would  also  prevent  detection  of  a  cuprous 
precipitate. 

228.  Test  the  latter  statement  as  follows  :  Make,  by  the  method 
previously  referred  to  (227),  a  mixture  of  alkali  hydroxid  con- 
taining Cupric  hydroxid.  Boil  the  mixture  and  notice  the  con- 
version of  the  blue  hydroxid  to  the  black  oxid.     Equation  : 

/OH 
Cu<        —  HjO  =  CuO 
\0H 

Cupric  by-  Cupric 

droxid  oxid 

(Insoluble:  black) 

These  difficulties  may  not  be  ignored  and  are  overcome  in  Fehl- 
ing's  test  (230),  which  can  be  better  understood  and  appreciated 
after  experience  with  Trommer's  test. 

229.  Repeat  test  227  with  a  solution  of  sodium  potassium  tar- 
trate (Rochelle  salt)  instead  of  sugar.  Notice  that  boiling  has  no 
effect  on  the  deep  blue  tartrate  solution.  Compare  these  effects 
with  those  of  the  preceding  experiment  and  also  with  those  already 
noticed  in  a  similar  experiment  with  glycerol  (i59)- 

230.  Fehling's  test  (L  :  226).  Mix  1  c.c.  each  of  the  "copper" 
and  "alkaline"  portions  of  Fehling's  solution.*     Boil  for  about  a 

*The  "copper"  and  "alkaline"  portions  of  Fehling's  solution,  as  prepared 
for  use,  were  made  as  follows  : 

^^  Copper"  portion.  34.64  grams  of  pure  crystalline  copper  sulfate  were  dis- 
solved in  500  c.c.  or"  distilled  water.  '■'Alkaline"  portion.  175  grams  of  pure 
crystalline  sodium  potassium  tartrate  ( Rochelle  salt)  and  50  grams  of  pure  sodium 
hydroxid  were  dissolved  in  500  c.c.  of  distilled  water. 

Fehling's  solution  is  a  mixture  of  equal  volumes  of  the  "copper"  and  "alka- 
line" solutions.  The  reactions  involved  in  its  preparation  are  indicated  by  the 
following  equations  : 

CuSO^    +     2K0H    =    Cu(0H)2     +     KjSO, 

Cupric  hy- 
droxid 
(Bluish-white : 
insoluble) 


Carbohydrates.  60 

minute.  No  change  in  the  color  of  the  solution  can  be  seen.*  Add 
an  equal  volume  of  liquid  containing  carbohydrate.  Boil  again  for  a 
minute  or  two.  A  greenish,  yellowish,  brownish  or  bright  red  color- 
ation results  according  to  the  degree  of  reduction,  the  proportion  of 
unchanged  copper  compound,  etc.,  as  was  previously  explained 
(227).     Let  the  mixture  stand  and  examine  the  deposit. 

The  chief  chemical  facts  involved  in  the  use  of  Fehling's  solution 
are  indicated  below  (see  footnote,  pages  68-69)  : 


•O— CH— COONa  Cu— OH         HO— CH— COOXa 

a<  I  +  R-CHO  +  2HsO  +  KOH=    |  +  2  |  H 

\0-CH— COOK  Cu— OH  HO— CH— COOK 


Cuprous 

hydroxid 

(Insoluble: 

yellow) 


Cu— OH  Cu. 

I     >0     +     H,0 


Cu— OH  Cu' 

Cuprous  oxid 
(Insoluble :  red) 

231.  Ny lander's  modification  of  the  Bbttger-Almen  test.  To  10 
parts  of  the  liquid  containing  the  carbohydrate  add  1  part  of  Ny- 
lander's  solution  f  and  boil  continuously  for  from  2  to  5  minutes. 

/OH  HO— CH— COONa  /O— CH— COONa  „^ 

Cu<  +  T  =     Cu<  +     2HsO 

\0H  HO— CH— COOK  ^O— CH— COOK 

Sodium  potassium  Sodium  potassium 

tartrate  cupro-tartrate 

(Deep  blue: 
soluble) 

Prepared  in  the  proportions  indicated  5  milligrams  of  glucose' will  reduce  all 
the  copper  compound  in  exactly  1  c.c.  of  Fehling's  solution  under  certain  fixed 
conditions.  The  solution  is  very  frequently  used  for  quantitative  determinations 
(235).  For  qualitative  purposes  the  solution  may  be  diluted  with  water  or  pref- 
erably with  hydroxid  solution  before  using. 

Fehling's  solution  readily  decomposes  soon  after  its  preparation,  in  which  condi- 
tion it  undergoes  spontaneous  reduction  on  boiling  and  cannot  be  relied  upon  for 
the  detection  of  reducing  substances.  By  keeping  the  "  copper  "  and  "alkaline" 
portions  in  separate  bottles,  however,  this  deterioration  is  prevented.  A  trifling 
quantity  of  phenol  or  other  "indifferent"  preservative  may  be  added  to  the 
"alkaline"  portion  to  prevent  development  of  fungi  and  any  decomposition  of 
the  alkaline  solution  that  might  possibly  occur  through  their  influence. 

*  If  a  change  occurs  the  solution  is  unfit  for  use. 

t  Nylander's  solution  is  usually  made  in  the  following  proportions  :  4  grams  of 
sodium  potassium  tartrate  ( Rochelle  salt)  are  dissolved  in  100  c.c.  of  5  per  cent, 
sodium  hydroxid  solution.  To  this  solution  2  grams  of  bismuth  subnitrate  are 
added  and  the  mixture  is  heated  on  a  water  bath  until  it  is  saturated  with  the 
bismuth  compound.     The  filtrate  is  the  reagent. 


70  Biochemical  Notes. 

If  reduction  occurs  the  solution  turns  yellow  at  first,  then  brown, 
and  finally  black.  On  standing,  a  black  precipitate  of  metallic 
bismuth,  or  of  a  mixture  of  bismuth  and  black  oxid  of  bismuth  is 
thrown  down. 

The  reactions  involved  in  the  use  of  Nylander's  solution  are  not 
definitely  known,  but  no  doubt  are  closely  analogous  to  those  indi- 
cated on  page  69  for  Fehling's  solution. 

232.  Barfoed^s  test.  To  about  5  c.c.  of  the  solution  add  a  few 
drops  of  Barfoed's  reagent.*  Heat  the  mixture  in  a  water  bath 
for  about  30  minutes.  Glucose  is  the  only  carbohydrate  that  will 
reduce  Barfoed's  reagent.  The  test  is  not  so  delicate  as  the  other 
reduction  tests.  The  reagent  soon  deteriorates  and  cannot  be  relied 
upon  unless  it  has  been  freshly  prepared. 

Demonstrations.  233.  Substances  and  conditions  that  inter- 
fere with  the  reduction  tests.  234.  Use  of  Haines'  and  Pavy's 
solutions.  235.  Quantitative  determination  of  sugar  with 
Fehling's  solution. 

236.  Percentage  amounts  of  carbohydrates  obtained  from 
some  mammalian  parts  and  from  various  vegetable  food-stuffs. t 

Fats  occur  in  relatively  greater  proportions  in  animals  than  in 
plants.  I     The  reverse  is  true  of  carbohydrates. 

The  figures  in  the  table  on  the  opposite  page  represent  general 
average  percentage  amounts  of  carbohydrate  matters  contained  in 
the  materials  named. 


*  Barfoed's  reagent  is  commonly  prepared  in  the  following  proportions :  20 
grams  of  cupric  acetate  are  dissolved  in  300  cc.  of  water.  To  this  solution  is 
added  10  c.c.  of  35  per  cent,  acetic  acid.  This  reagent  is  particularly  unlike  the 
other  copper  reagents  (Trommer's,  Fehling's,  Haines',  Pavy's)  in  being  acid  in 
reaction. 

t  The  proportions  of  carbohydrate  in  some  of  the  products  named  in  the  table 
on  the  opposite  page  vary  considerably  under  ordinary  conditions. 

X  The  largest  proportions  of  fats  in  plants  occur  in  the  so-called  oil-seeds.  In 
such  seeds  as  walnut  and  cocoanut  the  proportion  of  fat  in  the  true  seed-meat 
(endosperm)  amounts  to  from  30  to  40  per  cent,  of  the  fresh  material.  In  other 
vegetable  parts  the  proportional  content  of  fat  is  much  less,  e.  g.,  in  wood,  which 
contains  only  traces  (117). 


Carbohydrates.  71 

Animal  parts  *  (carbohydrate  is  chiefly  glycogen,  lactose  or 
glucose)  : 

Blood 0.1-0.15  (Glucose) 

Leucocytes  (thymus) 0.8  (Glycogen) 

Muscle 1.0-3.0      (Glycogen) 

Liver 1.5-4.5      (Glycogen) 

Milk 3.5-5.0      (Lactose) 

Urine  : 

Normal trace 

Abnormal  (diabetic)...  10  (or  more)  (Glucose)f 

Vegetable  parts  (carbohydrate  is  chiefly  starch  in  each  case) : 

Cucumber  2 

Cabbage 5 

Apple 13  j 

Potato 20 

Oat  (grains) 56 

Barley  (grains) 65 

Rye  (grains) 69 

Rice  (grains) 77 

237.  Phenylhydrazin  test.  This  is  one  of  the  most  important 
of  the  carbohydrate  tests.  It  has  already  been  stated  (page  50) 
that  the  raonosaccharids  contain  the  group, 

H 

H— (J— OH 

We  have  also  learned  that  phenylhydrazin  reacts  upon  aldehydes 
and  ketones,  uniting  directly  with  the  carbonyl  groups  as  follows 
(0  :  102)  '. 

R\       -.  R\ 


>C=:0  +    H,;N— NH-CgHs    =         >C=N— NH— CgHs  +  H^O 

W       W 


Aldehyde  Phenylhydrazin  Phenylhydrazon 

or  ketone 

Such  combinations  may  be  made  to  occur  between  phenylhydrazin 
and  the  carbohydrates  that  contain  free  carbonyl  groups.  But  in 
the  case  of  a  carbohydrate  that  reacts  with  phenylhydrazin,  the 

group,  HCOH,  which    is  attached  to  the  carbonyl  radical,  is  also 

*  Carbohydrate  occurs  universally  in  organisms.  In  nearly  all  animal  parts, 
however,  the  proportions  that  can  be  detected  are  extremely  slight. 

t  A  daily  elimination  by  diabetic  patients  of  2  pounds  or  more  of  glucose  baa 
been  observed  frequently. 

X  In  siccet  apples  most  of  the  starch  of  the  green  fruit  has  been  converted  into 
sucrose  by  the  ripening  process. 


72 


Biochemical  Notes. 


involved.  A  second  molecule  of  phenylhydrazin  withdraws  from 
that  group  the  two  hydrogen  atoms,  thus  converting  the  alcohol 
radical  into  a  carbonyl  group,  while  the  phenylhydrazin  itself  is 
converted  into  anilin  and  ammonia.  A  third  molecule  of  phenyl- 
hydrazin then  reacts  with  the  new  carbonyl  group  as  in  the  first 
case.  The  changes  in  the  natures  of  the  characteristic  groups  may 
be  indicated  as  follows  : 


HCOH 

Characteristic  group 
in  the  monosaccharids 


=N— NH— CfiHj 
=N-NH-aH, 


Characteristic  group  in  the 
di-phenylhydrazons,  or  osasones 


The  reactions  are  indicated  by  the  following  typical  equations  : 


H0=0 
HC— OH 

(CHOH); 

CH2OH 

Glucose 


+  H2N-NH-C6H5    = 


'^6^i 


+  H2O 


Phenylhydrazin 


HC==N— NH— CgHs 


O  iiHi 


(CH()H)3 
CH2OH 

Phenylglucoshydrazon 


>H,N-NH-C6H5- 
>  HjN— NH— CgHs- 


Phenylhydrazin 
(two  molecules) 


CH2OH 

Phenylglucoshydrazon 
(Soluble) 


,— >NH3  +  NH,-C6H5 
I    Ammonia        Anilin 

HC=N— NH— CeH^ 

"^     C=N— NH— CeHj    +  H^O 

(CHOH)^ 

CH2OH 

Phenylglucosazon 
(Insoluble) 


The  common  osazones  are  yellow  substances  that  may  be  crystal- 
lized readily.  Unlike  the  sugars,  they  are  only  slightly  soluble  in 
water  and  biological  liquids,  and  may  be  purified  without  difficulty 
by  recrystallization  from  various  solvents,  such  as  pyridin.  Each 
of  the  purified  products  has  a  characteristic  crystalline  appearance, 
melts  at  a  particular  temperature,  and  has  specific  optical  proper- 
ties. As  a  rule  the  identity  of  the  antecedent  sugar  may  be  readily 
ascertained  by  determinations  of  the  melting  point  of  the  purified 
osazon. 

The  insolubility  of  the  osazones  makes  it  an  easy  matter  to  pre- 
cipitate many  of  the  carbohydrates  from  mixed  solutions  containing 


Carbohydrates.  73 

other  substances.  The  corresponding  sugars  may  be  obtained  from 
the  osazones  by  various  methods.* 

238.  Apply  the  phenylhydrazin  test  as  follows  :  A.  Dissolve  in  a 
very  small  volume  of  water  a  mass  of  phenylhydrazin  hydrochlorid  f 
equal  to  that  of  a  pea.  Add  to  the  solution  about  the  same  bulk  of 
sodium  acetate.  Filter,  if  the  solution  does  not  get  clear  after 
repeated  agitation. 

B.  Pour  solution  A  into  about  5  c.c.  of  the  carbohydrate  liquid. 
Immerse  the  tube  in  boiling  water  and  keep  it  there  for  about 

*  "It  is  a  somewhat  remarkable  fact  that  methyl-phenylhydrazin, 
NH-N(CH3)-C6H5, 

yields  osazones  only  with  ketoses,  and  not  with  aldoses.  The  latter  form  color- 
less hydrazones  with  this  compound,  and  these  can  easily  be  separated  from  the 
intensely  yellow  osazones.  Methyl-phenylhydrazin  therefore  affords  a  valuable 
means  of  detecting  ketoses. 

"  When  the  osazones  are  carefully  warmed  with  hydrochloric  acid,  two  mole- 
cules of  phenylhydrazin  are  split  off,  with  formation  of  compounds,  osones,  con- 
taining two  carbonyl  groups.     In  this  way,  glucosazone  yields  glucosone, 

CHjOH— (CHOH)s— CO— HCO 

The  osones  can  be  reduced  by  treatment  with  zinc-dust  and  acetic  acid,  and  experi- 
ence has  shown  that  addition  of  hydrogen  always  takes  place  at  the  terminal 
C-atom.  GliTCOSone  yields  fructose,  CHjOH— (CHOH),— CO-CHjOH.  This 
reaction  affords  a  means  of  converting  aldoses  into  ketoses  : 

Aldose  s  >    Osazone  s  >   Osone  c  >    Ketose 

Inversely,  an  aldose  can  be  obtained  from  a  ketose.  On  reduction,  the  latter  yields 
a  hexahydric  alcohol,  which  is  converted  by  oxidation  into  a  monobasic  hexonic 
acid.  This  substance  splits  off  water,  yielding  the  corresponding  lactone,  which 
on  reduction  gives  the  aldose."     [Holleman.] 

Ketose  a->    Hexahydric  alcohol  = ->    Hexonic  acid  =  >    Lactone  s  >  Aldose 

Monosaccharids  may  be  transformed  directly  into  one  another  through  the  ac- 
tion of  very  dilute  alkalies.     Such  transformations  also  occur  in  the  body. 

t  Phenylhydrazin  is  a  liquid  base.  It  is  insoluble  in  water  and  ordinary  bio- 
logical liquids,  but  dissolves  readily  in  dilute  acids,  such  as  acetic  and  hydro- 
chloric. It  forms  soluble  salts  with  the  acids.  The  base  itself  cannot  be  used 
satisfactorily  in  the  tests  in  the  absence  of  acetic  acid.  As  a  rule  the  tests  are 
carried  out  with  the  solid  (and  soluble)  hydrochlorid,  C5H5 — NH — NH„  HCl .  The 
latter  substance  will  not  react  with  carbohydrates  in  the  absence  of  acetic  acid  or 
acetate.  In  the  tests  sodium  acetate  is  added  in  excess.  It  reacts  with  HCl  of  the 
hydrochlorid,  thus  producing  the  required  acetic  acid  and  also  sodium  chlorid  as 
a  by-product.  A  moderate  excess  of  the  reagent  is  essential  to  the  success  of  the 
test.  Failure  may  be  due  in  some  oases  to  formation  of  soluble  hydrazones  be- 
cause of  lack  of  suflacient  bydrazin. 


74  Biochemical  Notes. 

an  hour.  Finally  set  aside  the  tube  in  hot  water  in  a  beaker  and  let 
the  contents  of  the  tube  cool  slowly.  Examine  under  the  micro- 
scope any  deposit  that  may  be  formed. 

I.   Precipitation  from  Aqueous  Solutions. 

239.  The  carbohydrates  may  be  readily  precipitated  by  various 
reagents  from  their  aqueous  solutions.  Among  the  most  valuable 
precipitants  are  alcohol  (314)  and  ammonium  sulfate  (317). 

240.  Alcohol.  Transfer  to  small  beakers  10  c.c.  of  compara- 
tively concentrated  solutions  of  each  of  the  carbohydrates  under  ex- 
amination. Add  to  each  liquid  10  c.c.  of  95  per  cent,  alcohol. 
Stir  thoroughly.  Filter,  if  necessary,  on  dry  apparatus.  Test  the 
solubility  in  water  and  coloration  with  iodin  of  any  precipitate  that 
may  be  produced. 

241.  Repeat  the  additions  of  alcohol,  in  10  c.c.  portions  at  a 
time  to  each  beaker  (240),  until  a  total  of  50  c.c.  of  alcohol  has 
been  employed  in  this  way.  Stir  thoroughly  after  each  addition  of 
alcohol.  Filter  on  dry  apparatus  whenever  precipitation  is  induced, 
and  test  the  solubility  in  water  and  coloration  with  iodin,  of  any 
precipitate  that  may  be  produced. 

The  more  complex  the  carbohydrate  the  more  readily  it  may  be 
precipitated  by  alcohol.  Relatively  large  proportions  of  alcohol 
are  required  to  precipitate  monosaccharids.  All  polysaccharids  are 
easily  precipitated  completely  from  their  aqueous  solutions  by 
alcohol. 

242.  Remove  the  alcohol  from  each  of  the  final  filtrates  or 
unprecipitated  liquids  (241),  by  evaporation  on  a  water  bath.  Test 
the  concentrated  liquids  with  iodin  and  Fehling's  solution. 

243.  Ammonium  sulfate.  Transfer  to  small  beakers  12  c.c.  of 
comparatively  concentrated  solutions  of  each  of  the  carbohydrates 
under  examination.  Follow  the  plan  of  the  experiments  with  alco- 
hol, but  use,  instead  of  alcohol,  ammonium  sulfate  as  follows  : 

244.  A.  Half-saturation  with  ammonium  sulfate.  Treat  10  c.c. 
of  the  aqueous  carbohydrate  solution  with  10  c.c.  of  saturated 
aqueous  solution  of  ammonium  sulfate.  Stir  thoroughly.  Let  the 
mixtures  stand  about  10  minutes.  Filter  if  necessary,  on  dry 
apparatus.  Determine  the  solubility  in  water  of  any  precipitate 
that  may  be  formed ;  also  the  iodin  reaction  of  the  product. 


Carbohydrates.  75 

245.  B.  Saturation  with  ammonium  sulfate.  Treat  with  iodin 
or  Feliling's  solution  a  small  portion  of  each  of  the  unprecipitatod 
liquids,  or  any  filtrates  that  may  have  been  obtained  (244).  To  the 
main  bulk  of  the  carbohydrate  solution  add  powdered  ammonium 
sulfate  until  the  liquid  is  saturated  at  room  temperature.  Avoid 
excess  of  sulfate.  Filter.  Apply  to  the  filtrates  the  tests  indicated 
in  section  244. 

Common  starches  are  of  two  kinds  :  (a)  ordinary  starch,  which  is 
insoluble  in  cold  water  and  of  which  practically  all  the  undis- 
solved matter  in  starch  paste  is  composed ;  (6)  "  soluble  "  starch  or 
"amidulin,"  which  may  be  formed  from  insoluble  starch  by  initial 
hydration,  is  produced  in  this  way  in  starch  paste,  and  is  contained 
in  the  filtrate  from  starch  paste.  Each  of  the  starches  yields  a 
blue  compound  with  iodin,  that  with  insoluble  starch  failing  to 
dissolve  in  water,  whereas  the  product  with  soluble  starch  dissolves 
readily  in  water.  ,  The  viscid  masses  of  insoluble  starch  in  starch 
paste  are  immediately  dehydrated  and  made  flocculent  by  half-satu- 
ration of  the  paste  with  ammonium  sulfate.  Soluble  starch  is  com- 
pletely precipitated  in  24  hours,  only  incompletely  in  a  few  min- 
utes, by  half  saturation  of  its  solution  with  ammonium  sulfate. 

Dextrins  are  of  two  general  types :  («)  erythrodextrins  (I-III), 
which  yield  soluble  reddish  compounds  with  iodin,  and  (6)  achroo- 
dextrins,  which  fail  to  yield  colored  compounds  with  iodin.  Of  the 
erythrodextrins,  1  and  II  are  completely  precipitated  by  saturation 
of  their  solutions  with  ammonium  sulfate ;  erythrodextrin  III  and 
achroodextrins  are  not  precipitated  under  such  conditions. 

The  sugars  are  not  precipitated  from  aqueous  solutions  by  ammo- 
nium sulfate. 

The  iodin  colorations  obtained  Avith  the  erythrodextrins  are  the 
following :  Erythrodextrin  I,  purple ;  erythrodextrin  II,  red ; 
erythrodextrin  III,  reddish  brown.  Erythrodextrin  I  is  com- 
pletely precipitated  from  aqueous  solution  by  saturation  with  mag- 
nesium sulfate ;  erythrodextrin  II  is  not  precipitated  under  such 
conditions. 

Demonstrations.  246.  Various  additional  methods  of  pre- 
cipitating carbohydrates.  247.  Detection  of  carbohydrate  in 
the  presence  of  fat,  fatty  acid,  soap  and  glycerol. 


76  Biochemical  Notes. 


J.  Hydration  of  Carbohydrates  (170). 

248.  Monosaccharids  may  be  obtained  by  hydration  from  all 
other  classes  of  carbohydrates  and  from  carbohydrate-yielding  sub- 
stances, such  as  glucosids  (185)  and  various  proteins  (250). 

249.  Place  in  small  beakers  20  c.c.  of  each  of  the  carbohydrate 
solutions.  Add  to  each  solution  an  equal  volume  of  0.2  per  cent, 
hydrochloric  acid.  Cover  the  beakers  with  watch  glasses.  Boil 
each  solution  gently  about  30  minutes.  Finally  neutralize  the 
solutions  and  apply  to  each  tests  223,  230  and  232. 

K.   Relationship  Between   Carbohydrate    and   Protein. 

250.  Most  of  the  proteins  (252)  respond  positively  to  Molisch's 
test  (222).  Some  proteins  may  be  made  to  yield  more  or  less  car- 
bohydrate material  on  chemical  decomposition.  In  plants  carbo- 
hydrates are  combined  with  various  amino  substances  (264)  to  form 
proteins.  Among  the  transformation  products  of  proteins  in  organ- 
isms are  carbohydrates.  Some  of  the  proteins  may  be  regarded  as 
complex  glucosids  (184).  A  few  of  the  proteins  contain  carbohy- 
drate radicals  in  so  large  proportions  and  yield  so  much  carbohydrate, 
material  on  decomposition  that,  as  a  group,  they  are  called  gluco- 
proteins  (255).  The  characters  of  the  various  carbohydrate  radicals 
that  are  certainly  present  in  many  proteins  have  not  been  satisfac- 
torily determined,  but  among  the  known  hydration  products  are 
glucosamin  (177)  and  galactosamin.  The  latter  is  a  stereoisomer 
of  the  former  (171). 

These  facts  lead  to  the  deduction  that  carbohydrate  is  utilized 
directly  or  indirectly  in  organisms  for  the  synthesis  of  some  perhaps 
of  all  kinds  of  proteins. 


CHAPTER   IV. 
PROTEINS.* 

A.    Demonstration  of  Protein  Products. 

251.  Representatives  of  the  groups  of  proteins  named  in  the 
summary  on  pages  79-80. 

B.   General  Characteristics  of  Proteins. 

252.  Distribution.  "  Few  substances  are  so  widely  distributed 
in  nature  as  proteins  and  certainly  none  are  of  more  consequence 
from  a  biological  point  of  view.  The  tissues  of  all  plants  and 
animals  contain  these  substances  in  large  proportions  and  of  the  in- 
variable organic  constituents  of  every  living  functionally  active 
cell  the  albuminous  (protein)  are  undoubtedly  the  most  important. 

253.  Molecular  complexity.  ''That  the  proteins  are  among 
the  most  complex  compounds  with  which  the  chemist  has  to  deal, 
and  therefore,  also,  the  most  elusive  in  chemical  research,  are  de- 
ductions to  which  the  experiences  of  all  protein  investigators  seem 
to  point  conclusively.  In  spite  of  the  fact,  however,  that  the 
proteins  have  long  been  the  subjects  of  persistent  and  carefully 
conducted  chemical  investigation,  our  knowledge  of  the  molecular 
configuration  of  the  proteins  still  remains  decidedly  indefinite  and 
all  attempts  thus  far  completely  to  unravel  the  constitution  of  the 
protein  molecule  have  resulted  negatively.  Each  of  the  various 
theories  which  have  been  proposed  in  regard  to  structural  formulas 
depends  chiefly  upon  the  nature  of  the  products  obtained  by  protein 
decompositions,  since  all  of  the  numerous  attempts  to  prepare  time 
albuminous  material  artificially  have  invariably  resulted  in  failure. 
Since  the  decomposition  products  of  protein  matter  are  multitudinous 
and,  under  different  conditions  so  various,  it  is  not  at  all  difficult  to 

*The  words  "protein"  and  "proteid  "  are  synonymons  in  English,  although 
the  latter  term  is  gradually  becoming  obsolete.  The  phrase  "albuminous  sub- 
stance "  is  also  frequently  used  synonymously  in  English.  In  German  "  Pro- 
teid  "  and  the  equivalent  of  "  albuminous  substance  "  ( "  Eiweisskorper  " )  are 
used  to  designate  sub-groups  of  "  proteins  "  ( "  Proteine  "). 

77 


78  Biochemical,  Notes, 

comprehend  why  the  biological  chemist  is  so  much  in  the  dark  as 
to  the  real  configuration  of  the  protein  molecule  and  why,  in  the 
absence  of  sufficient  data  afforded  by  artificial  synthesis,  he  is  able  to 
form  only  hypotheses  as  to  the  manner  in  which  the  analytic  nuclei 
obtained  from  proteins  are  held  in  the  undecomposed  substances. 

254.  Synthesis  in  organisms.  "  Although  true  protein  matter 
has  never  been  prepared  in  the  laboratory  from  any  of  its  decomposi- 
tion products,  its  synthesis  is  constantly  taking  place  in  plants,  and 
to  a  certain  extent,  in  animals  as  well.  The  ultimate  origin  of  pro- 
teins may  be  traced  to  the  vegetable  kingdom,  however,  for  plants 
are  constantly  transforming  inorganic  matter  into  albuminous  sub- 
stances, as  a  part  of  the  process  of  their  development,  whereas  in 
animal  metabolism,  albuminous  syntheses  are  wholly  dependent 
upon  protein  derivatives  that  are  assimilated  after  digestion  of  pro- 
tein food."  * 

C.   Classification  of  Proteins. 

255.  Groups.  The  fats  and  carbohydrates  may  be  satisfactorily 
classified  on  the  basis  of  intramolecular  differences.  Such  a  chem- 
ical classification  cannot,  however,  be  made  of  proteins  at  present, 
because  of  the  meagerness  of  our  knowledge  of  the  intramolecular 
nature  of  these  very  complex  substances.  The  empirical  classifica- 
tion of  proteins  that  is  suggested  on  pages  79  and  80  depends,  in 
the  main,  upon  superficial  distinctions,  such  as  similarity  or  dis- 
similarity in  solubility,  precipitability,  derivability  from  more  com- 
plex proteins,  convertibility  into  simpler  proteins  and  so  on.  The 
chief  merits  of  the  classification  proposed  are  its  convenience,  and 
the  general  physical,  chemical  and  biological  relationships  that  it 
makes  evident. f 

*Gies  :  Yale  Scientific  Monthly,  1898,  iv,  pp.  204-205  ;  also,  Gies  and  collab- 
orators, Biochemical  Researches,  1903,  i,  pp.  719-720  (Reprint  No.  39). 

Although  the  remarks  that  are  quoted  above  (252-254)  were  written  by  the 
author  nearly  ten  years  ago,  they  require  very  little  qualification  now,  in  spite  of 
the  fact  that  many  admirable  investigations  have  been  carried  out  since  then  in 
order  if  possible  to  throw  more  light  on  the  molecular  nature  of  protein  matter. 
This  fact  emphasizes  the  diflQculties  of  the  problems  indicated. 

t  The  difficulties  involved  in  perfecting  a  classification  of  the  proteins  are  so 
great  that  each  investigator  of  the  protein  substances  is  apt  to  have  a  classification 
of  his  own.  For  this  reason  the  student  who  uses  this  volume  is  advised  to  consult 
other  authors  on  this  subject. 


Proteins.  79 

Classification  of  Proteins  (255).* 

I.  Primary  proteins  or  true  albuminous  substances. 

A.  Proteins  that  occur  in  organisms  as  free  compounds,  as  simple 

salts  of  the  common  inorganic  ions  (or  molecules),  or  as  sim- 
ple organic  compounds,  and  which  cannot  as  a  rule  be  ob- 
tained unchanged  from  other  proteins  by  laboratory  methods. 

a.  Albumins  —  Cell  albumins,  sey^um  albumins. 

b.  Globulins  —  Cell  globulins,  myosin,  fibrinogen,  edestin. 

c.  Phosphoglobulins —  Caseinogen,  mucous  'protein. 

B.  Proteins  that  occur  naturally  only  in  combination  with  other 

substances  (in  compound  secondary  proteins)  but  which  may 
be  obtained  from  the  latter  by  appropriate  laboratory  methods. 

a.  Histons  —  Globin,  leucocyte  histon^  spermatozoan  histon. 

b.  Protamins  —  Clupein,  salmin,  scombrin,  sturin. 

II.  Secondary  proteins  or  derivatives  of  true  albu- 

minous substances. 
A.    Compound  derivatives. 

a.  Natural  compound  proteins :  derivatives  or  compounds  of 

true  albuminous  substances  that  cannot  be  exactly  dupli- 
cated by  known  laboratory  methods  and  which  occur  in 
organisms  free  or  as  salts  of  common  inorganic  ions  or 
molecules. 

1.  Nucleoproteins  —  Nucleohiston,  cytoglobin. 

2.  Chromoproteins  —  Hemoglobin,  hemocyanin. 

3.  Glucoproteins : 

Non-phosphorized  —  Osseomucoid,  salivary  mucin. 
Phosphorized  —  Ichthulin,  helicoprotein. 

4.  Lecithalbumins. 

5.  lodoproteins  —  Thyreoglobulin. 

b.  Artificial  compound  proteins  :  additive  products  which  re- 

sult from  combinations  of  various  kinds  of  proteins  with 
other  proteins  (or  non-protein  organic  substances)  that 
may  be  brought  about  by  laboratory  methods. 
1.  Similar  to   natural   compound  proteins.  —  Albumin 
combined  with  glucoproteins,  lecithin  and  various  other 
colloidal  substances  that  normally  occur  in  organisms. 
3.  Special  organic  salts.  —  Albumin  combined  with  color- 
ing matters,  picric  acid  and  other  organic  reagents. 
*Only  oommou  members  of  each  group  are  named. 


80  Biochemical  Notes. 

S.  Simple  derivatives. 

a.  Simple  derivatives    of  true   albuminous    substauces,  that 

cannot  he  made  by  laboratory  methods  and  which  occur 
chiefly  in  the  tissues  as  structural  materials  ;  they  appear 
to  be  condensation  products  and  are  often  called  * '  albu- 
minoids." Chief  among  them  are,  (1)  collagen,  (2) 
elastin,  (3)  albumoid,  (4)  keratin. 

b.  Simple  proteins  that  are  made   in  organisms  from  other 

proteins  and  may  also  be  made  from  other  proteins  by 
laboratory  methods. 

1.  From  proteins  in  general. 

i.  Proteoses  —  Albumose,  caseose. 
ii.  Peptones  —  Globulin  pepton,  myosin  pepton. 

2.  From  most  primary  and  compound  proteins,  and  from 

groups  4  (II,  B,  b)  and  C  (II)  of  simple  derivatives. 
Proteinates  —  Acidalbumin,  alkali  albuminate. 

3.  From  collagen  only — Gelatin. 

4.  Solidified  by  enzymes. 

i.   Casein  (from  caseinogen). 
ii.  Fibrin  (from  fibrinogen), 
iii.  Plasteins  (from  proteoses), 
iv.  Myosin  fibrin  (from  myosin). 
C.  Proteins  that  do  not  occur  normally  in  organisms,  hut  which 
may  he  made  in  the  laboratory,  from  various  primary  and 
secondary  proteins. 
a.  Coagulated  proteins — Produced  by  heat,  alcohol  or  other 

means,  such  as  coagulated  albumin, 
h.  Special  inorganic  salts  of  proteins —  Coppet-  albuminate, 
globulin  phosphotungstate. 
III.  Digestive,  also  arti£cial  synthetic  products,  that 
resemble  in  some  respects  a  few  of  the  simplest 
proteins  —  Peptids. 
256.   Group  properties.    The  properties  characteristic  of  the  sub- 
groups of  proteins  that  are  indicated  on  pages  79  and  80  are  so  varied 
and  the  peculiarities  of  the  individual  members  of  each  group  are  so 
marked  that  we  shall  find  it  convenient  to  delay  further  consideration 
of  the  differences  among  the  proteins  until  we  have  become  more 
familiar  with  their  general  properties  as  exhibited  by  the  various  typ- 
ical proteins  selected  for  the  tests  (269).     In  our  study  of  the  tissues 
and  body  fluids  each  of  the  important  proteins  will  be  considered 
from  this  standpoint  in  connection  with  its  situation  and  functions. 


Proteins. 


81 


D.    Elementary  Composition. 

257.  Qualitative  elementary  composition.  Like  fats  and  carbo- 
hydrates, all  protein  molecules  contain  carbon,  hydrogen  and  oxygen. 
But  all  protein  molecules  are  unlike  those  of  fats  and  carbohydrates 
in  containing  nitrogen,  besides.  Most  protein  molecules  also  contain 
sulfur ;  many  contain  phosphorus  in  addition  and  some  likewise  con- 
tain iron,  copper,  iodin  or  other  unusual  elements  in  strict  "  organic 
combination."  Protein  salts,  many  of  which  occur  naturally,  con- 
tain various  elements  in  addition  to  those  just  mentioned. 

258.  Typical  formulas.     The  following  empirical  formulas  of 

three  typical  proteins  show  at  a  glance  the  diversity  and  complexity 

so  characteristic  of  the  proteins  as  a  group  : 

Pepton C,iH3,N«09 

Serum  albumin C45oH72oH„60i4oS6 

Hemoglobin ^iti^vioz^iTfiiK^-^^ 

259.  Quantitative  elementary  composition.  The  figures  for 
percentage  elementary  composition  of  some  of  the  more  important 
proteins,  arranged  in  the  order  of  classification  (pages  79  and  80), 
are  summarized  below  : 


Serum  albumin 

Egg  albumin 

Serum  globulin 

Edestin 

Fibri  nogen 

Leucocyte  histon  (Thymus). 

Globin 

Clupein 

Pancreatic  Nucleoprotein 

Thymus  Nucleohistoc 

Hemoglobin  

Hemocyanin  * 

Osaeomucoid , 

Ichthulin 

Thyreoglobulin  f  • 


Elastin 54.14 


Chondroalbumoid 

Fibrinoses  J  : 

T,  •  f  Proto 

Primary...  ^jj^^^^^ 

(A  (Thio).. 
Secondary  ■{  i?  (Gluco). 

{c     

Antipepton 

Gelatin 

Casein 

Fibrin 

Coagulated  egg  albumin.. 


c 

H 

N 

53.08 

7.10 

15.93 

52.75 

7.10 

15.51 

52.71 

7.01 

15.85 

51.27 

6.85 

18.76 

52.93 

6.90 

16.66 

52.37 

7.70 

18.35 

54.97 

7.20 

16.89 

47.24 

8.14 

25. 7  J 

51.35 

6.81 

17.82 

48.80 

7.03 

18.37 

54.57 

7.22 

16.38 

53.66 

7.33 

16.09 

47.07 

6.69 

11.98 

53.52 

7.71 

15.64 

51.85 

6.88 

15.49 

54.14 

7.33 

16.87 

50.46 

7.05 

14.95 

55.12 

6.61 

17.98 

55.64 

6.80 

17.66 

48.96 

6.90 

16.02 

48.72 

7.03 

13.76 

34.52 

5.85 

17.24 

46.20 

6.74 

16.26 

50.11 

6.56 

17.81 

53.07 

7.13 

15.64 

52.68 

6.83 

16.91 

52.33 

6.98 

15.84 

o 


21.99 
22.90 
23.32 
22.22 
22.26 
20.96 
20.52 
18.90 
20.93 
21.59 
20.43 
21.67 
31.85 
22.19 
23.57 
21.52 
25.68 


1.90 
1.62 
1.11 
0.91 
1.25 
0,62 
0.42 

i.29 
0.51 
0.57 
0.86 
2.41 
0.41 
1.87 
0.14 
1.86 


19.07 

18.69 

25.15 
30.49 
42.89 

30,80  I  ... 

25.24 

22.60 

22.48 

23.04 


1.22 
1.21 
2.97 


0.26 
0.76 
1.10 
1.81 


1.67 
3.70 


0.43 


0.80 


Fe 


0.13 
6.34 

6.16 


*  Content  of  copper  =  0.38  per  cent. 
X  Compare  -with  the  figures  for  fibrin. 


t  Content  of  iodin  =  0.34  per  cent. 


82 


Biochemical  Notes. 


E.  Cleavage  Products  of  Proteins. 
260.  It  is  obvious  that  the  figui'es  given  on  page  81  for  ele- 
mentary composition  of  proteins  tell  very  little  about  the  molecular 
nature  of  the  products  referred  to.  Thus  the  figures  for  percentage 
elementary  composition  of  egg  albumin  and  serum  globulin  are 
practically  the  same  : 


C 

H 

N 

0 

S 

Eee  albumin 

52.75 
52.71 

7.10 
7.01 

15.51 
15.85 

22.90 
23.32 

1.62 

Serum  globulin 

1.11 

These  proteins  are  decidedly  different  in  certain  respects,  as  we 
shall  see,  yet  the  figures  for  composition  suggest  that  they  are  prac- 
tically the  same.  From  this  standpoint  these  two  proteins  are 
similar  to  isomers  such  as  the  aldohexoses  (171),  which,  although 
exactly  the  same  in  elementary  composition  are  very  different  in 
certain  respects  because  of  their  unlikenesses  in  molecular  con- 
figuration. 

Chemists  have  long  appreciated  the  fact  that  a  more  perfect 
knowledge  of  protein  matter  awaits  complete  determinations  of  the 
internal  structure  of  protein  molecules.  But  many  of  the  difficul- 
ties of  such  determinations  have  been  insuperable. 

261.  As  a  rule  the  molecular  construction  of  a  compound  is  as- 
certained satisfactorily  by  two  general  methods  of  investigation  : 

1.  The  substance  under  examination  is  subjected  to  cleavage 
(analysis)  and  the  characters  of  the  decomposition  products  are 
carefully  ascertained. 

2.  The  cleavage  products  are  reunited  by  appropriate  methods 
(synthesis)  and  the  original  substance  is  thus  regenerated. 

When  these  two  processes  give  complete  and  perfectly  satisfac- 
tory results  the  intramolecular  qualities  of  the  substance  under  ex- 
amination may  be  accurately  established.  Both  of  these  methods 
of  investigation  have  been  applied  to  proteins,  but  thus  far  the  list 
of  cleavage  (analytic)  products  that  may  be  obtained  from  proteins 
is  far  from  complete  and  no  one  has  succeeded  in  putting  more 
than  a  few  of  the  very  many  cleavage  products  together  again.  As 
has  already  been  indicated  (page  81),  the  imperfect  synthetic  prod- 
ucts that  have  been  made  resemble  some  of  the  simplest  proteins, 
but  as  yet  they  are  unlike  all  of  the  proteins  in  very  important 


Pkoteins.  83 

respects.  This  synthetic  success,  incomplete  though  it  has  been, 
makes  it  appear  to  be  only  a  question  of  time  and  investigation, 
however,  until  natural  proteins  can  be  perfectly  synthesized  from 
their  cleavage  products,  and  until  typical  proteins  can  be  made  in 
the  laboratory  almost  as  readily  as  typical  sugars  can  now  be  pro- 
duced synthetically.  These  are  no  reasons  for  thinking  that  these 
desirable  results  are  unattainable. 

The  known  cleavage  products  of  the  various  proteins  are  quali- 
tatively much  the  same.  The  widest  variations  from  the  average 
qualitative  results  afforded  by  any  given  method  of  cleavage  are  ex- 
hibited by  the  compound  proteins,  which  yield  cleavage  products 
that  represent  not  only  the  strictly  protein  part  of  their  molecules, 
but  also  the  non-protein  portion. 

The  qualities  of  the  cleavage  products  of  proteins  vary  consider- 
ably also  with  the  characters  of  the  methods  employed  to  effect 
decomposition.  The  molecules  are  so  intricate  in  structure  that, 
speaking  figuratively,  the  fragments  which  result  from  breaking 
the  molecule  to  pieces  vary  in  size,  shape  and  other  characters  as 
the  force  of  the  blow  that  is  administered  to  the  molecule  is  in- 
creased or  decreased,  or  as  the  blow  falls  upon  one  pointer  another 
on  the  surface  of  the  molecule. 

As  has  already  been  indicated  most  of  the  proteins  may  be  con- 
verted by  laboratory  methods  into  other  proteins,  which  are  usually 
only  of  a  simple  type,  however,  such  as  proteinates,  proteoses  and 
peptones.  Such  protein  products  tiiat  result  from  the  modification 
of  more  complex  proteins  need  not  be  considered  here. 

262.  In  the  following  summary  a  few  of  the  common  methods 
that  are  used  to  effect  profound  decomposition  of  proteins  are  men- 
tioned with  the  names  of  the  more  important  non-protein  cleavage 
products  that  are  obtainable  from  typical  proteins  by  the  processes 
indicated. 

Igyiition.  —  Water,  carbon  dioxid,  ammonia,  hydrogen  sulfid,  in- 
flammable gases,  nitrogenized  bases,  etc. 

Fusion  with  caustic  alkali.  —  Ammonia,  carbon  dioxid,  methyl 
mercaptan,  indol,  skatol,  phenol,  fatty  acids  (salts)  such  as  acetic, 
butyric  and  valeric,  leucin,  tyrosin,  etc. 

Putrefactio7i.  —  Fatty  acids,  phenol  and  indol  derivatives,  pto- 
mains,  carbon  dioxid,  hydrogen  sulfid,  hydrogen,  ammonia,  methane, 
methyl  mercaptan,  etc. 


84  Biochemical  Notes. 

The  decomposition  products  obtained  by  the  three  methods  just 
indicated  result  from  very  profound  disorganization  of  the  carbon 
nuclei  or  chains  of  the  protein  molecules  and  on  that  account  do 
not  indicate  very  much  as  to  the  inner  structure  of  the  molecules 
from  which  they  have  been  derived.  By  these  drastic  methods,  the 
structural  elements  are  altered  beyond  recognition. 

263.  The  most  significant  non-protein  cleavage  products  of  pro- 
teins are  those  obtained  by  hydrolysis  with  boiling  dilute  mineral 
acids  or  through  the  agency  of  certain  proteolytic  enzymes  that  may 
be  extracted  from  organisms.  These  methods  of  cleavage  are  less 
drastic  than  the  three  named  above.  Consequently  the  resultant 
segments  of  the  carbon  chains  that  were  originally  present  in  the 
molecule  are  longer  so  to  speak,  also  less  altered,  and  more  indica- 
tive of  the  way  in  which  the  chains  are  joined  together  in  the 
molecule.  These  relatively  mild  methods  of  cleavage  also  seem  to 
exert  less  influence  on  the  bonds  between  the  carbon  and  nitrogen 
atoms,  and  the  cleavage  products  that  are  obtained  with  the  aid  of 
these  methods  are  more  like  simple,  unmodified  fragments  than 
those  produced  by  the  action  of  the  more  vigorous  means  of  effect- 
ing decomposition. 

264.  Hydrolysis.  The  most  important  cleavage  products  that 
result  from  hydration  of  practically  all  true  proteins  through  the 
action  of  boiling  dilute  mineral  acids  may  be  classified  as  follows  : 

Aliphatic  Products. 
Nitrogenous,  free  from  sulfur. 

Mono-basic,  mon-amino  acids. 

Amino-acetic  (glycocoU),  CH2<^ 

^COOH 

a- Amino-propionic  (alanin),  CH3 — CH<' 

^000  H 

?^  /NH, 

a- Amino-/3-oxy -propionic  (serin),    CH2 — CH<( 

\COOH 

a-Amino-»-valeriCj  CH, — CHj — CHg — CH<' 

^COOH 

CH3V  /NH, 

a-Amino-iso-bntyl-acetic  (leucin),  /CH — CH, — CH<r^ 

CH/  \C0OH 


Proteins.  85 

Di-basic,  mon-amino  acids. 

COOH 
Amino-succinic  (aspartic  acid),        |  /NH, 

CHj— CH< 

^COOH 


COOH 

a-Amino-slutaric  (glutamic  acid),   T  /NH^ 

CH— CH2-CH< 

^COOH 

Mono-basic  di-amino  acid. 

/NH, 
a,  5-Di-amino-n-valeric  (ornithin),  CHj<(  /NH, 

\CHj— CH,-CH< 

\COOH 
Hexon  bases. 

oi-amino-/3-imidazol  propionic  acid  (histidin), 
/NH, 


HC=C— CHj— CHC 

[         ]  ^COOH 

HN        N 


H 

Guanidin-a-amino-n-valeric  acid  (arginin), 
/NH, 
HN=C<  /NH, 

\nH— CH,— CH,— CHj— CH< 

XJOGH 
a,  c-Di-amino-n-caproic  acid  (lysin), 
/NH, 
Ch/  .NH, 

XiJHo    CH»    CHq    CHv 

\C00H 

Carbohydrate  derivative. 
Glucosamin  (178). 

Nitrogenous,  containing  sulfur. 

CH,— SH 
o-Amino-/3-tbio-lactic  acid  (cystein),  CH — NH, 


COOH 

CH, — S S — CH, 


o-Di-amino-/3-di-i;hio-di-lactylic  acid  (cystin),  CH — NH,  CH — NH, 

COOH  COOH 

Sulfurous,  free  from  nitrogen. 

:h. 


a-Thio-lactic  acid,  CH— SH 

COOH 

Ethyl  Bulfid,  >S 

c,h/ 

Mercaptans,   e.  j.,  methyl  meroaptan,    CH3 — SH 


86  Biochemical,  Notes. 

Caebocyclic  Products. 

Phenyl-a-amino-propionic  acid  (phenyl-alaniu),  CH — NHj 

COOH 
CH^— CeU^— OH 

jj-Oxy-phenyl-a-amino-propionic  acid  (tyrosin),  CH — MHg 

COOH 

Heterocyclic  Products    (see  hexon  bases). 
Pyrrol  derivatives. 

CH, — CH, 

)         I 
a-Pyrrolidin-carboxylio  acid  (prolin),  CH^    CH — COOH 

NH 

CH,— CH— OH 

I  i 

Oxy-pyrrolidin-a-carboxylic  acid,  CHj    CH — COOH 

NH 

Indol  derivative.  ch^— nh^ 

C— CH<' 
Indol-/3-amino-propionic  acid  (tryptophan),  CgHX    ^CH 

NH 

265.  Relations  of  cleavage  products  to  molecular  structure. — 

So  far  as  our  present  knowledge  extends  we  may  say  that  the  sub- 
stances named  on  pages  84—86  are  the  most  significant  cleavage 
products  of  proteins.  As  a  rule,  over  50  per  cent,  of  the  nitrogen 
of  proteins  may  be  obtained  by  hydrolysis  in  the  form  of  mon- 
amino  acids.  When  proteins  are  treated  with  nitrous  acid  only, 
relatively  slight  proportions  of  nitrogen  are  evolved.  If  amino 
groups  were  contained  in  proteins  to  the  extent  that  the  large  yield 
of  mon-amino  acids  might  be  assumed  to  indicate,  a  correspondingly 
large  proportion  of  nitrogen  would  be  evolved  as  a  result  of  the 
aforesaid  treatment  with  nitrous  acid  (0  :   184). 

It  must  be  distinctly  understood  that  none  of  the  above-named 
products  exists  as  such  in  the  protein  molecule,  but  rather  that  the 
molecular  nucleus  of  each  particular  cleavage  product  is  probably 
combined  with  other  such  nuclei  to  form  the  whole  protein  molecu- 
lar frame-work.  It  is  quite  probable  also  that  the  products  obtained 
by  hydration  are  merely  fragments  of  larger  intra-protein  nuclei  and 
that  the  particular  products  of  hydrolytic  cleavage  vary  in  nature 


Proteins.  87 

with  differences  in  the  characters  of  attached  groups  in  the  various 
proteins.  It  is  generally  believed  that  the  precursors  in  the  pro- 
tein molecule  of  the  amino  groups  in  the  amino-acid  cleavage  prod- 
ucts are  mainly  imino  groups  (0  :  i8l)  and  that  the  latter  are 
converted  into  amino  radicals  by  the  hydration  process. 

Most  of  the  proteins  yield  on  hydrolysis  practically  all  of  the 
cleavage  products  already  noted.  Other  proteins  have  failed  to 
yield  some  of  the  products.  No  two  kinds  of  proteins  yield  the 
same  proportions  of  any  of  the  cleavage  products. 

F.    Constitution  of  Proteins  and  Attempts  at  Synthesis. 
266.  Peptids.     All  of  the  amino-acid  cleavage  products  of  pro- 
teins (pages  84-86)  are  a-amino-acids  (0:  184)  and  each  is  char- 
acterized by  containing  the  group 

H 

— CH<  or    HjN— C— COOH 

^COOH  I 

The  a-amino-acids  may  readily  be  made  to  unite  with  one 
another,  in  which  process  the  amino  group  of  one  molecule  unites 
with  the  carboxyl  group  of  the  other,  with  elimination  of  water,  as 
is  indicated  by  the  following  typical  equation  : 

: ■;;;::::h; 

H,N— CHj— C0|0H;+HN— CHj— COOH  =  H^N— CH,— COiNII— CHj— COOH 

Glvcocoll  Glycyl-glycin 

(two  molecules)  (one  molecule) 

Glycyl-glycin  is  both  an  amid  (0  :  184)  and  an  amino-acid,  and  is 
a  member  of  the  group  of  substances  called  di-peptids.  Similar 
amid-ami no-acids  have  been  obtained,  among  which  the  substances 
with  the  following  formulas  are  now  well  known  : 

Di-peptids : 

A lanyl-alanin,  H^N— CH— COjNH— CH— COOH 

CH,  CH3 

Leucyl-leucin,  H^N— CH— COjNH— CH— COOII 

Tri-peptid : 

Di-glycyl-glycin,  H^N— CH,— COiXH- CH  — COiNfl— CHj-  COOH 

Tetra-peptid : 

Tri-glycyl-glycin, 
H»N— CH,— COiNH— CH,— COiNH— CH,— CoiNH— CH,— COOH 


88  Biochemical  Notes. 

267.  Peptid  chains  in  protein  molecules.  —  The  complex  pep- 
tids  resemble  in  some  respects  a  few  of  the  simplest  proteins  such 
as  peptones.  This  synthetic  approximation  to  some  natural  pro- 
teins has  led  to  the  belief  that  the  peptid  chain  is  part  of  the 
structure  of  the  protein  molecule  and  that  the  proteiu  molecule 
contains  a  skeleton  comprising  parts  such  as  are  represented  by  the 
following  chain-fragment : 

— iNH— CB— COjNH— CH— COiNH— CH— CoInH— CH— COjNH— CH— COr- 
E  E  E  K  E 

The  following  formula  suggests  in  a  general  way  the  manner  in 
which  some  of  the  many  known  cleavage  products  of  proteins  may 
be  related  to  each  other  in  protein  molecules  : 

-^NH-CH-COjNH-CH-COjNH-CH-CO:NH-CH-COjNH CH-COJ- 


CH3  C.Hg  C,Hj  OH,  C 

COOH  CsH.OH    HN<^C-CH3 

CgH^ 

(Alanin)  (Leucin)  (Glutamic  (Tyrosin)  (Tryptophan*) 

acid) 

There  is  no  reason  to  believe,  however,  that  the  protein  molecule 
is  a  simple  chain  such  as  is  indicated  by  the  above  formula.  It  is 
probable  that  such  chains  are  interlinked  in  very  complex  ways 
and  also  that,  when  the  molecule  is  cut  up  by  a  process  of  hydra- 
tion, the  various  portions  of  the  interlinked  segments  are  converted 
into  the  nuclei  of  the  various  amino-acids  named  on  pages  84—86. 

The  structure  that  is  indicated  by  the  above  formulas  is  at  most 
only  the  approximate  structure  of  portions  of  protein  molecules,  but 
it  enables  us  to  understand  why  it  is  that,  although  the  true  pro- 
teins yield  so  many  diflPerent  cleavage  products,  they  are  neverthe- 
less so  much  the  same  in  their  general  properties. 

268.  Every  protein  is  both  polybasic  and  polyacidic.  —  The 
protein  molecule  consists  so  largely  of  amino-acid  nuclei  that  pro- 
teins themselves  show  some  of  the  general  properties  of  amino- 
acids.  An  amino-acid  contains  both  a  carboxyl  group  and  an 
amino  group,  and  will  form  salts  readily  with  both  acids  and  bases. 
Consequently  an  amino-acid  is  both  acidic  and  basic  in  its  qualities. 
But  the  carboxyl  and  amino  groups  counterbalance  each  other  in 
these  respects  and  amino-acids  are  practically  neutral  in  reaction. 

The  relations  between  a  typical  amino-acid  and  forms  of  the  two 

*  Written  as  skatol-amino-acetic  acid.     See  page  86. 


Proteins.  89 

general  kinds  of  its  salts  may  be  summarized  in  connection  with 
amino-acetic  acid  in  aqueous  solution  as  follows  : 

Glycocoll:  H^N— CH^— COOH.  Practically  neutral.  Disso- 
ciation very  slight. 

Glycocoll  hydrochlorid  :  HCl,  H^N— CH^— COOH.  Dissociated 
into  the  neutral  compound  and  into  H*  and  CI'  ions.     Strongly  add. 

Sodium  glycocollate  :  HgN — CH^ — COONa.  Dissociated  into  the 
neutral  compound  and  into  Na*  and  OH'  ions.     Strongly  alkaline. 

Proteins,  by  reason  of  their  araino-acid  properties  are  at  once 
weak  acids  and  weak  bases.  Every  protein  combines  with  bases 
and  acids  to  form  readily  dissociable  salts.  Some  proteins  are 
more  acid  than  basic,  or  vice  versa.  The  glucoproteins  represent 
the  former  kind,  the  protamins  the  latter.  All  proteins  are  both 
polybasic  and  polyacidic  because  of  the  many  amino-acid  nuclei  they 
contain.  These  facts  account  for  the  variety  of  combinations  into 
which  proteins  enter  in  organisms  and  in  the  laboratory  (313-321). 

G.   Typical  Proteins  for  Use  in  the  Tests. 

269.  Kinds.  In  experiments  280-328  use  solid  samples  or  the 
specially  prepared  solutions  of  each  of  the  following  typical  pro- 
teins :  albumin  (in  white  of  egg),  edestin  (from  hempseed),  add- 
albumin  (from  meat)  and  proteoses  (Witte's  —  from  fibrin).  Protein 
properties  that  cannot  be  learned  from  a  study  of  these  products 
will  be  noted  during  the  progress  of  our  tissue  studies. 

270.  Preparation.  Crude  albumin.  Strike  an  egg  sharply  on 
the  edge  of  a  casserole  with  sufficient  force  to  cut  the  egg  almost  in 
two.  Turn  the  severed  egg  on  end,  hinge  back  the  upper  portion 
and  remove  the  white  matter  to  the  casserole  by  transferring  the 
yolk  back  and  forth  carefully  from  one  portion  of  the  shell  to 
the  other. 

271.  Temporary  disposition  of  the  yolk.  Transfer  the  yolk  to 
the  large  stoppered  bottle.  Add  to  it  about  100  c.c.  of  alcohol. 
Stir  the  mixture  thoroughly  and  set  it  aside  for  future  use. 

272.  Dry  egg  white.  With  scissors  cut  up  the  membranes  in  the 
white  matter  (270).  Transfer  about  half  the  material  to  a  porcelain 
dish  and  evaporate  it  to  dryness  on  a  water  bath  at  a  temperature 
not  above  45°  C.  Stir  repeatedly  to  favor  desiccation.  After  the 
material  has  been  dried  pulverize  and  bottle  it  for  future  use. 


90  Biochemical  Notes. 

273.  Crude  albumin  solution.  Egg  white  contains  about  12  per 
cent,  of  protein  substances.  Of  these  albumin  is  predominant  and 
the  characteristic  "  albuminous  "  properties  of  egg  white  are  due  to 
it.*  The  associated  substances  in  egg  white  do  not  interfere  with 
a  study  of  the  properties  of  the  albumin. 

274.  Prepare  a  crude  albumin  solution  by  treating  the  remain- 
ing portion  of  egg  white  as  follows  :  Ascertain  the  volume  of  the 
available  supply  of  egg  white  (272)  and  transfer  it  to  a  flask.  Add 
to  it  about  10  volumes  of  water.  Shake  the  mixture  thoroughly 
and  pass  the  solution  through  a  wet  folded  filter.  The  filtrate  is 
ready  for  use. 

275.  Edesiin.  Mix  in  a  beaker  10  grams  of  ground  hempseed 
and  100  c.c.  of  5  per  cent,  sodium  chlorid  solution  heated  to  60°  C. 
Stir  the  mixture  thoroughly.  Place  the  beaker  in  hot  water  in  a 
water  bath  and  maintain,  for  about  30  minutes,  a  temperature  of 
55°  to  60°  C.  The  temperature  must  not  be  permitted  to  go  above 
60°  C.  After  such  treatment  for  30  minutes  filter  the  mixture  on 
a  filtration  apparatus  wetted  with  5  per  cent,  solution  of  sodium 
chlorid.  Catch  the  filtrate  in  a  large  beaker  immersed  in  hot 
water  (60°  C.)  in  a  casserole  or  water  bath.  After  the  filtrate  has 
been  collected,  heat  it  on  a  water  bath  to  60°  C.  and  add  to  it  suffi- 
cient warm  water  (60°  C.)  to  render  the  liquid /am%  turbid.  Cover 
the  beaker  v/ith  a  watch  glass  and  set  it  aside  in  hot  water  (60°  C.) 
so  that  the  extract  may  cool  as  slowly  as  possible. 

276.  Edestin  solution.  After  the  diluted  extract  has  cooled 
(275)  decant  and  filter  the  supernatant  liquid,  which  is  saturated  at 
room  temperature  with  edestin  and  may  be  regarded  as  a  crude 
edestin  solution.  As  in  the  case  of  the  albumin  solution,  the  asso- 
ciated constituents  do  not  prevent  determination  of  the  general 
properties  of  the  edestin.     Reserve  the  filtrate  for  use  in  the  tests. 

277.  Crystalline  edestin.  Examine  under  a  microscope  the 
white  sediment  obtained  from  the  hemp  seed  extract  (276).  It 
consists  wholly  of  edestin,  which  is  usually  entirely  crystalline  — 
octahedra  mainly.  Filter  the  sedimentary  mixture.  Wash  the 
precipitate  with  water  from  the  paper  into  the  original  beaker. 
Add  a  large  volume  of  water  to  the  mixture,  and  stir  thoroughly. 

*  The  generic  term  that  is  used  by  the  Germans  to  designate  the  primary  or 
true  proteins  is  Eiweisskorper,  i.  e.,  egg-white  substances  (page  77). 


Proteins.  91 

After  sedimentation,  decant  the  supernatant  wash  water,  filter  off 
the  solid  matter  and  wash  the  latter  until  it  is  practically  free  from 
chlorid.     Use  the  moist  precipitate  in  the  tests. 

278.  Acidalbumin.  Place  about  10  grams  of  hashed  meat  in  a 
large  beaker  full  of  water  and  stir  thoroughly  to  remove  blood  and 
soluble  matter.  Repeat  the  process  several  times  until  the  decanted 
liquid  is  free  from  coloring  matter.  Transfer  the  washed  meat  to  a 
small  beaker  and  treat  it  with  about  100  c.c.  of  0.2  per  cent,  hydro- 
chloric acid.  Heat  as  high  as  possible  on  a  water  bath.  The  acid 
mixture  should  be  stirred  repeatedly.  After  heating  for  about  15 
minutes  filter  the  mixture  and  neutralize  approximately  with  dilute 
potassium  hydroxid  solution.  A  precipitate  of  acidalbumin  will  be 
obtained  as  the  reaction  of  the  liquid  approaches  the  neutral  point. 
Filter  the  mixture,  transfer  the  precipitate  to  a  large  amount  of 
water  in  a  beaker,  stir  thoroughly  so  as  to  wash  off  traces  of  ad- 
herent acid  or  alkali  and,  after  sedimentation,  decant  and  filter  again. 
Wash  the  product  on  the  paper  until  the  washings  are  neutral. 

279.  Proteose.  Use  "  Witte's  pepton,"  a  commercial  product, 
consisting  chiefly  of  proteoses  with  some  pepton,  obtained  from  fibrin 
by  hydration  methods  similar  to  that  shown  in  demonstration  290. 

H.   Detection  of  the  Elements  Contained 
IN  Proteins  (206). 

280.  Apply  tests  50,  51,  53,  62,  63  and  64  to  a  sample  of  one 
of  the  protein  products. 

I.    Physical  Properties  of  Proteins. 

281.  Proteins,  like  carbohydrates,  differ  considerably  both  physi- 
cally and  chemically. 

282.  Proteins  are  nonvolatile  and  do  not  impart  greasy  stains 
to  paper  (208). 

283.  Most  of  the  pure  proteins  are  neutral  compounds  (209). 

284.  Microscopic  appearance  (210). 

285.  Solubilities  (211). 

286.  Proteins  do  not  form  emulsions  (102,  212). 
Demonstrations.     287.  Separation   of   albumin  in  quantity 

from  egg  white  and  its  purification  by  dialysis.  288.  Prepar- 
ation of  crystalline  egg  albumin.  289.  Preparation  of  gelat- 
inous proteinate.  290.  Hydration  of  primary  protein  succes- 
sively to  simple  secondary  protein  and  to  amino-acids,  with 


92  Biochemical  Notes. 

methods  for  the  identification  of  some  of  the  latter.  291. 
Optical  properties  of  proteins.  292.  Ignition  of  proteins,  with 
an  examination  of  the  products  of  destructive  distillation. 
293.  Putrefaction  of  proteins,  with  an  examination  of  the  prod- 
ucts. 294.  Effects  on  proteins  of  various  kinds  of  bacteria. 
295.  Tests  of  the  diffusibility  of  proteins.  296.  Frothiness  of 
protein  solutions. 

J.    Color  Tests. 

297.  Lack  of  specificity  of  protein  reactions.  All  proteins 
respond  in  common  to  certain  chemical  tests.  None  of  the  so-called 
protein  tests  is  strictly  characteristic  of  protein  matter,  however, 
for  each  of  the  reactions  is  given  by  at  least  one  non-protein 
substance.  These  remarks  apply  not  only  to  color  tests  but  to  all 
others  in  which  proteins  may  be  involved.  The  very  great  com- 
plexity of  the  protein  molecule  and  the  diversity  of  its  side  chains 
and  interlocked  nuclei  account  for  the  remarkable  manner  in  which 
proteins  share  with  various  other  substances  certain  important 
reactions. 

As  a  rule  any  non-protein  substance  that  behaves  exactly  like 
protein  in  a  given  test  fails  to  simulate  protein  in  any  other  special 
reaction.  On  this  account  it  is  possible,  through  the  evidence  of  a 
number  of  corroboratory  tests,  to  determine  definitely  whether  or 
not  protein  is  contained  in  a  given  medium,  and  also  to  exclude  the 
influence  of  non-protein  substances.  The  effects  of  non-protein  sub- 
stances on  protein  reactions  have  been  pretty  thoroughly  studied. 

The  color  reactions  that  are  given  by  proteins  are  given  by  par- 
ticular nuclei  contained  in  the  proteins.  A  reaction  that  is  given 
by  a  particular  nucleus  in  protein  matter  is  given  as  a  rule  by  any 
other  substance  having  the  same  nucleus. 

The  following  special  coloration  tests  (298-307)  are  given  by 
practically  all  proteins  in  solid  form  or  in  solution. 

Biuret  test  (L:  252).*  298.  A.  To  about  5  c.c.  of  the  solu- 
tion in  a  porcelain  evaporation  dish  f  add  sufficient  potassium 
hydroxid  to  make  the  solution  strongly  alkaline.  Do  not  heat  the 
mixture  for  the  reason  that  some  proteins  are  completely  decom- 
posed by  heat  in  such  alkaline  solutions. 

*  Also  occasionally  called  Piotrowski's  test.     It  was  discovered  by  Rose  in  1833. 

t  The  porcelain  affords  a  white  background  and  makes  it  possible  to  detect  the 
very  slight  coloration  that  results  when  only  traces  of  proteins  are  present.  Prac- 
tically the  same  result  is  obtained  by  making  the  test  in  a  beaker  resting  on  dry 
white  filter  paper. 


Proteins.  93 

299.  B.  Prepare  a  very  dilute  cupric  sulfate  solution  by  adding 
about  2  drops  of  2  per  cent,  solution  of  cupric  sulfate  to  a  common 
test-tube  full  of  water.  The  solution  should  be  practically  color- 
less. An  excess  of  cupric  sulfate  may  obscure  the  characteristic 
color  of  the  test  (227). 

300.  C.  Pour  the  copper  solution  (B)  gradually  into  the  alka- 
line solution  (A).  If  protein  is  present  a  bluish  violet  to  reddish 
violet  or  a  bright  red  or  pink  coloration  results. 

301.  If  the  result  was  positive  notice  whether  a  stronger  cupric 
sulfate  solution  intensifies  the  color. 

The  biuret  reaction  is  given   by  all  substances  that  contain  two 

—  CONH,  groups  united  together  or  attached  to  a  nitrogen  atom 

or  to  a  carbon  atom.     Among  such  substances  are  the  following  : 

.CONHj  /L'ONHj  CONIIj 

\cONH2  '    ^CONH,  CON^L 

Biuret  Malonamid  Oxamid 

That  protein  will  yield  biuret  or  a  biuret-like  substance  on 
cleavage  will  seem  apparent  after  comparison  of  the  above  formulas 
with  those  on  page  88  which  show  the  amino-acid  nature  of  parts  of 
the  protein  molecule. 

The  color  of  the  test  is  due  to  the  formation  of  biuret  potassium 
cupric  hydroxid.  The  equation  may  be  represented  as  a  reaction 
between  biuret  and  the  reagents  used,  as  follows  : 

OH  HO 

2  >NH-f-CnSO,4.4KOH    =  >NH  HN\^_^  +  KjSO^ 

0=cC  0=Q<  >C-0 

OH  HO 

Biuret  Biuret  potassium  cupric  hydroxid 

(soluble :  red) 

Precautions.  Strongly  acid  solutions  should  be  approximately 
neutralized  before  the  biuret  test  is  applied  to  them,  otherwise  the 
final  alkalinity  may  not  be  sufficient  to  effect  transformation  of  the 
protein  into  the  colored  compound. 

The  test  cannot  be  applied  in  the  presence  of  relatively  large 
proportions  of  substances  that  react  with  alkali  hydroxid,  such  as 
magnesium  sulfate  and  ammonium  sulfate.  Their  removal  is  nec- 
essary before  the  addition  of  cupric  sulfate  (225). 


94  Biochemical  Notes. 

302.  Xanthoproteic  test.*  To  about  3  c.c.  of  the  neutral  or 
slightly  acid  solution  in  a  test-tube  add  approximately  3  c.c.  of  con- 
centrated nitric  acid.  If  protein  is  present  the  solution  will  be 
colored  slightly  yellowish.  Some  proteins  are  precipitated  by  nitric 
acid,  but  their  precipitates  gradually  dissolve,  becoming  yellowish 
in  the  process  and  yielding  yellow  matter  to  the  solution.  Boil  for 
a  minute.  •  The  yellow  color  is  increased  to  canary  yellow  if  a 
moderate  amount  of  protein  is  present.  If  a  protein  precipitate 
was  formed  when  the  acid  was  added  to  the  solution,  the  precipitate 
will  be  made  more  yellowish  by  the  higher  temperature  and  will  be 
perceptibly  decreased  in  quantity  because  of  hastened  solution. 

Cool  the  acid  solution  in  running  water.  Pour  gently  down  the 
side  of  the  tube  after  cooling,  f  a  moderate  excess  of  ammonium 
hydroxid.  If  protein  was  present  the  color  of  the  alkaline  liquid 
along  the  line  of  junction  of  the  two  solutions  will  be  deepened  to 
orange.  Shake  the  mixture  and  thus  increase  the  depth  of  the  layer 
in  which  ammonium  hydroxid  has  overcome  the  acid.  The  orange 
band  is  thereby  increased  in  width  and  if  ammonium  hydroxid  is 
present  in  sufficient  excess,  the  entire  solution  on  complete  mixture 
will  be  suiFused  by  an  orange  color. 

The  reaction  is  given  by  many  substances,  among  them  aromatic 
compounds  in  general.  The  coloration  is  due  to  the  formation  of 
various  indeterminate  aromatic  nitro-derivatives.  The  aromatic 
nuclei  in  the  protein  molecule  that  are  chiefly  responsible  for  the 
coloration  seem  to  be  those  of  tyrosin  and  tryptophan  (264). 

303.  Millon's  test.  Treat  about  3  c.c.  of  the  solution  in  a  test 
tube  with  approximately  3  c.c.  of  Millon's  reagent.  J  If  protein  is 
present  a  white  precipitate  may  be  formed  or  precipitation  may  fail 
to  occur,  according  to  the  nature  of  the  contained  protein.  Heat 
the  mixture  gently  and  carry  the  temperature  slowly  to  the  boiling 

*  Among  the  nitroderivatives  of  protein  matter  produced  by  treatment  with 
concentrated  nitric  acid  is  a  substance  or  a  mixture  of  several  substances  called 
xanthoprotein.  Very  little  is  known  about  this  product.  The  test  was  discov- 
ered by  Fourcroy  and  Vauquelin  in  1805. 

t  Hot  nitric  acid  and  ammonium  hydroxid  react  violently. 

X  Millon's  reagent  is  made  as  follows  :  Mercury  is  dissolved  in  its  own  weight 
of  pure  nitric  acid  (sp.  gr.  1.4).  This  concentrated  solution  is  treated  with  two 
volumes  of  water  and  the  diluted  mixture  is  allowed  to  stand  24  hours.  A  slight 
sediment  of  basic  salts  will  form.  The  filtrate,  containing  mercurous  and  mer- 
curic nitrates,  excess  of  nitric  acid  and  a  small  amount  of  nitrous  acid,  is  the 
reagent. 


Proteins.  95 

point.  If  all  of  any  protein  present  has  been  precipitated,  the 
precipitate  will  assume  a  red  color  as  the  temperature  rises  and  the 
solution  itself  will  be  colorless.  The  precipitate  may  dissolve  and 
color  the  solution  red.  If  unprecipitated  protein  was  present,  the 
solution  itself  will  be  reddened. 

The  reaction  is  given  by  all  aromatic  substances  that  contain  a 
hydroxyl  radical  attached  to  a  benzol  ring.  Thus,  it  is  given  by 
phenol,  Cgll^  -  OH,  but  not  by  benzoic  acid,  CgH^  —  COOH. 
The  aromatic  nucleus  in  protein  matter  that  seems  to  be  respon- 
sible for  the  coloration  is  oxy-phenyl-a-amino-propionic  acid  (ty- 
rosin,  264).     The  nature  of  the  red  compound  is  unknown. 

Precautions.  Excessive  heating  should  be  avoided  as  a  rule. 
A  white  precipitate  is  given  by  urea,  sulfates  and  other  non- pro- 
tein substances.  Alkaline  solutions  should  be  acidified  slightly 
with  nitric  acid  before  Millon's  reagent  is  added  to  them,  otherwise 
oxids  of  mercury  may  be  precipitated  from  the  reagent  by  the  alkali 
and  the  reagent  rendered  valueless.  Inorganic  salts  when  present 
in  excess  also  interfere  with  the  reaction. 

304.  Hopkins  and  Cole's  modification  of  Adamkiewicz'  test, 
or  the  glyoxylic  test.  To  about  2  c.c.  of  the  solution  add  an 
equal  volume  of  "reduced  oxalic  acid  solution."*  Mix  thor- 
oughly. Pour  gently  down  the  side  of  the  tube  an  equal  volume 
of  concentrated  sulfuric  acid.  If  protein  is  present  a  band  of 
purple  will  form  along  the  line  of  junction  of  the  two  solutions. 
Shake  gently  in  order  to  mix  the  two  solutions  gradually.  If  pro- 
tein is  present  the  entire  liquid  will  be  colored  purple  on  mixing  the 
solutions  uniformly. 

This  is  a  test  for  tryptophan  (264)  and  is  due  to  the  tryptophan 
nucleus  in  the  protein  molecule.  The  chemical  character  of  the 
colored  product  is  unknown. 

Precautions.  The  reaction  is  prevented  by  an  excess  of  chlorids 
and  by  certain  salts  that  are  not  conspicuous  in  biological  liquids, 
such  as  nitrates.  Various  carbohydrates,  such  as  sucrose,  are  con- 
verted into  black  or  colored  products  by  concentrated  sulfuric  acid 

*  "  •  Reduced  oxalic  acid '  is  prepared  by  treating  half  a  liter  of  a  saturated  solu- 
tion of  oxalic  acid  with  40  grams  of  2  per  cent,  sodium  amalgam  in  a  tall  cyl- 
inder. When  all  the  hydrogen  has  been  evolved  the  solution  is  filtered  and 
diluted  with  twice  its  volume  of  water.  The  solution  now  contains  oxalic  acid, 
sodium  binoxalate,  and  glyoxylic  acid  (COOH— CHO).  It  should  be  kept  in  a 
closed  bottle  containing  a  little  chloroform."     [Cole.] 


96  Biochemical  Notes. 

and  they  accordingly  interfere  with  the  test  by  obscuring  the  char- 
acteristic color.  Pure  concentrated  sulfuric  acid  is  essential  — 
some  common  impurities  in  ordinary  commercial  sulfuric  acid  may 
prevent  the  reaction. 

305.  Liebermann's  test  (310).  Treat  a  small  amount  of  solid 
protein  matter  with  a  moderate  quantity  of  alcohol  and  heat  the  mix- 
ture for  about  1 5  minutes  on  a  water  bath  to  eifect  thorough  dehydra- 
tion of  the  protein.  Transfer  the  protein  matter,  after  filtration, 
to  a  small  amount  of  ether  in  a  test-tube  for  removal  of  adherent  fat. 
Shake  repeatedly.  After  exposure  to  the  ether  for  about  an  hour  or 
more,  transfer  the  dry  protein  to  a  small  beaker,  add  to  it  a  moderate 
quantity  of  concentrated  hydrochloric  acid  and  boil  vigorously  for 
several  minutes.  The  protein  dissolves.  A  deep  blue  or  violet 
blue  is  the  characteristic  color  of  the  test.  The  nature  of  the  col- 
ored substance  is  unknown. 

The  reaction  is  regarded  as  a  furol  test  and  is  thought  to  be  due 
to  the  simultaneous  presence  in  the  molecule  of  a  carbohydrate  nu- 
cleus and  an  oxy-phenyl  group.  It  may  also  be  due  to  tryptophan 
in  reaction  with  glyoxylic  acid  (304)  from  the  ether  employed. 

306.  Molisch's  test.  Apply  the  test  as  suggested  in  section 
222.  A  positive  result  indicates  the  presence  of  a  carbohydrate 
nucleus. 

307.  Test  for  loosely  combined  sulfur  (58,  59,  55).  A  posi- 
tive result  depends  upon  the  production  of  hydrogen  sulfid  from 
mercaptan  groups  in  the  molecule.  The  mercaptan  groups  are  con- 
tained in  the  nuclei  of  sulfurous  cleavage  products,  such  as  cystin 
and  others  referred  to  on  page  85. 

Demonstrations.  308.  Conditions  that  interfere  with  the 
color  reactions  of  proteins.  309.  Effects  of  iodin  on  proteins 
(115,  218).  310.  Effects  of  cold  and  hot  concentrated  mineral 
and  organic  acids  and  alkalies  on  proteins  (315).  311.  Produc- 
tion of  carbohydrate  material  from  a  protein  (tendomucoid). 
312.  Precipitation  of  proteins  by  agitation. 

K.  Precipitation  of  Proteins. 
313.  We  have  already  learned  that  in  the  free  state  practically 
all  proteins  are  neutral  compounds.  We  have  also  noted  the  fact 
that,  like  amino-acids,  all  proteins  are  at  once  polyacid  and  poly- 
basic  in  the  presence  of  acids  and  bases  (268).  In  the  free  state 
most  of  the  proteins  dissolve  in  water  without  undergoing  hydro- 


Peoteins.  97 

lytic  dissociation.  Water  is  the  most  general  protein  solvent.  In 
the  presence  of  acids  and  bases,  i.  e.,  ions,  the  dissolved  protein 
is  ionized  and  it  forms  salts  that  may  be  soluble  or  insoluble  ac- 
cording to  the  nature  and  effects  of  the  non-protein  ions.  Pro- 
teins may,  therefore,  be  anions  or  cations  according  to  the  electrical 
conditions  of  the  ions  that  are  introduced  into  solution  with  them. 
Determine  the  precipitative  effects  upon  protein  solutions  of  the 
reagents  indicated  below. 

314.  Alcohol.  (240).  Alcohol  is  one  of  the  most  general  of 
protein  precipitants.  All  proteins  are  insoluble  in  absolute  alcohol. 
Some  proteins  are  soluble  in  95  per  cent,  alcohol,  however,  and  a 
greater  number  are  soluble  in  more  dilute  alcohol.  Consequently 
some  proteins  are  difficult  to  precipitate  from  aqueous  solution  with 
alcohol.  The  minimal  degree  of  concentration  for  precipitation 
differs  greatly  for  the  various  groups  of  proteins. 

The  precipitate  that  is  thrown  from  aqueous  protein  solutions  by 
alcohol  is  the  protein  itself  as  it  existed  in  solution.  The  alcohol 
does  not  combine  with  it.  Some  proteins  are  quickly  rendered  in- 
soluble by  alcohol,  after  their  precipitation,  probably  by  a  dehydra- 
tion process.  Other  proteins  do  not  seem  to  be  affected  chemically 
in  any  way  by  alcohol  under  such  conditions. 

315.  Strong  mineral  acids  —  Nitric,  hydrochloric,  ml/uric. 
Pour  protein  solutions  gently  down  the  sides  of  the  tubes  upon  the 
acids  in  order  to  make  "  ring  tests."  Notice  the  white  precipitates 
that  may  be  produced.  Shake  the  mixtures  gently  to  effect  com- 
plete mixture  gradually. 

316.  Notice  the  effects  of  excessive  additions  of  the  acids  (315). 
Boil  half  of  each  solution.  Set  aside  the  boiled  and  the  cold  portions 
for  later  observation  of  the  colorific  effects  (310).  The  production  of 
various  colors  from  the  same  colorless  material  by  different  agents 
suggests  the  same  possibility  in  organisms.  Many  of  the  coloring 
matters  in  organisms  are  biological  cleavage  products  of  proteins. 

317.  Saturation  with  neutral  salts  —  Sodium  chlorid,  mag- 
nesium sulfate,  ammonium  sulfate  (243).  Add  the  finely  powdered 
salts  to  small  quantities  of  protein  solutions  in  beakers.  Avoid  un- 
necessary excesses  of  the  salts.  Afler  each  solution  has  been  satur- 
ated, pour  it  into  a  narrow  test-tube  and  notice  whether  a  protein 
precipitate  has  been  produced. 


98  Biochemical  Notes. 

318.  Acidify  with  acetic  acid  any  of  the  solutions  that  may  not 
have  been  precipitated  by  saturation  with  a  salt  (317).  Notice 
that  acetic  acid  alone  will  not  precipitate  to  the  same  degree,  if  at 
all,  any  of  the  original  protein  solutions. 

319.  Filter  any  of  the  solutions  that  may  have  been  precipitated 
by  saturation  with  a  salt  (317)  and  apply  to  the  filtrate  in  each 
case  the  biuret  reaction  (301). 

Ammonium  sulfate,  when  added  to  full  saturation  of  their 
aqueous  solutions,  completely  precipitates  all  forms  of  protein 
matter  except  peptones  and  some  proteoses  (243). 

The  protein  precipitates  that  are  produced  by  saturation  with 
neutral  salts  are  protein-saline  compounds. 

The  effects  just  noted  are  comparable  to  the  process  of  "  salting 
out"  soaps  (129)  and  polysaccharids  (245). 

320.  Salts  of  heavy  metals  —  Silver  nitrate,  mercuric  chlorid, 
plumbic  acetate,  cupric  sulfate,  ferric  chlorid.  Notice  the  effects  of 
moderate  proportions  and  of  excesses  of  the  reagents. 

Each  precipitate  consists  of  a  compound  of  a  metallic  cation  and 
a  protein  anion.  Cations  of  the  heavy  metals  completely  precipi- 
tate most  of  the  proteins  from  neutral,  acid  and  alkaline  solutions. 

321.  Alkaloidal  reagents  —  Phosphotungstic  acid,  tannic  acid, 
picric  acid,  trichloracetic  acid,  potassio-mercuric  iodid,  potassium 
chromate,  hydroferrocyanic  acid.  Apply  the  reagents  after  acidi- 
fication of  the  protein  solution  with  dilute  hydrochloric  acid.  In 
the  last  test  (hydroferrocyanic  acid),  acidify  the  solution  with  acetic 
acid  and  add  potassium  ferrocyanid.  Hydroferrocyanic  acid  results  * 
and  immediately  acts  upon  the  protein. 

Each  precipitate  consists  of  a  compound  of  a  reagent  anion  and 
a  protein  cation.  Anions  of  the  alkaloidal  reagents  completely 
precipitate  most  of  the  proteins  in  acid  solutions.  In  neutral  or 
alkaline  solutions  protein  compounds  with  the  alkaloidal  reagents 
are,  as  a  rule,  completely  dissociated  and  remain  in  solution.  Bases 
that  are  as  weak  as  most  of  the  proteins  do  not  form  precipitates 
with  these  reagents  in  the  absence  of  an  excess  of  H*  cations. 

*  Equation  :    K,Fe(CN")6  +  4CB3COOH  =  H,Fe(CN)6  +  4CH3COOK 
Potassium  Hydroferro- 

ferrocyanid  cyanic  acid 


Proteins.  99 

L.    Coagulation  of  Protp:ins. 

322.  Albumins,  globulins,  proteinates,  and  a  few  of  the  secon- 
dary (compound)  proteins  that  contain  albumin-  or  globulin-like 
nuclei,  may  be  rendered  completely  insoluble,  i.  e,,  coagulated,  if 
they  are  heated  while  dissolved  or  suspended  in  neutral  or  faintly 
acid  liquids.  The  high  temperature  causes  a  permanent  chemical 
alteration  which  is  not  yet  understood.  In  the  coagulation  process 
a  very  slight  proportion  of  alkaline  reacting  material  (NH^?)  is 
thrown  out  of  the  molecule,  intramolecular  rearrangement  occurs, 
and  in  neutral  or  faintly  acid  liquids  all  of  the  coagulable  protein 
separates  as  a  flocculent  precipitate  (coagulum).  The  weight  of  the 
coagulum,  in  carefully  conducted  experiments,  is  practically  the 
same  as  that  of  the  original  protein  (330).  There  is  no  way  by 
which  the  coagulum  can  be  reconverted  into  the  original  protein. 
Coagulation  is  a  physical  expression  of  a  permanent  though  rela- 
tively slight  chemical  alteration.  Coagulable  proteins  have  specific 
coagulation  temperatures. 

323.  Coagulation  of  solid  primary  proteins.  Suspend  in  water 
or  10  per  cent,  salt  solution  albumin,  globulin,  or  acidalbumin. 
Boil  the  mixture  for  a  minute  or  two.  Filter.  Observe  that  the 
solid  substance  no  longer  dissolves  in  some  of  the  reagents  that  had 
previously  been  found  to  dissolve  it  readily,  e.  g.,  0.2  per  cent, 
hydrochloric  acid  (285). 

324.  Coagulation  of  dissolved  primary  proteins.  To  about  10 
c.c.  of  clear  neutral  solutions  of  albumin  (HgO)  and  edestin  (5  per 
cent.  NaCl)  in  small  beakers  add  20  c.c.  of  water.  Boil.  Observe 
the  opalescence  that  is  produced  as  the  boiling  points  are  neared. 
Test  the  reactions  of  the  hot  liquids.*  Add  to  each  several  drops 
of  very  dilute  acetic  acid.f  Notice  the  immediate  flocculation  that 
results.  Again  test  the  reaction  of  each  liquid.  Add  excess  of 
dilute  acid,  e.  g.,  0.2  per  cent,  hydrochloric  acid  (328). 

Non-coagulability  of  proteinates  in  acid  or  alkaline  solvents 


*  In  the  coagulation  process  a  very  slight  amount  of  alkaline  reacting  material 
is  produced  from  the  proteid  (322).  It  may  be  too  slight  for  detection  with  an 
ordinary  indicator,  without  special  treatment  of  the  liquid. 

t  Prepare  very  dilute  acetic  acid  by  allowing  a  single  drop  of  common  36  per 
cent,  acetic  acid  to  fall  into  an  ordinary  test-tube  full  of  water.  Shake  thoroughly. 
Use  this  very  dilute  solution  in  the  test  referred  to  above. 


100  Biochemical  Notes. 

and  also  of  proteoses  (representing  groups  A,  b,  i  and  3  of  the 
simple  secondary  proteins).  325.  Repeat  the  last  experiment 
(324)  with  acid  (0.2  per  cent.  HCl)  or  alkaline  (0.5  per  cent.  NagCOg) 
solutions  of  acidalbumin. 

326.  Repeat  with  proteose  experiments  323  and  324. 
Determinations  of  temperatures  of  coagulation  and  of  the 

effects  on  coagulability  (and  its  temperatures)  of  the  (A)  con- 
centration of  the  protein,  of  the  (B)  reaction  of  the  solvent  and 
of  (C)  associated  saline  matter. 

327.  Prepare  in  narrow  test-tubes  of  equal  size  the  following 
solutions  :  * 

A .  Influence  of  concentration  of  the  protein. 

1.  5   c.c.   of  neutral  dilute  albumin  solution   (1  part  of  egg 

white  +39  parts  of  water). 

2.  5  cc.  of  neutral  concentrated  albumin   solution  (1   part  of 

egg  white  -}-  9  parts  of  water). 

3.  Solution  2  -f  5  c.c.  of  water  (1  part  of  egg  white  in  20). 

B.  Influence  of  the  reaction  of  the  solvent. 

4.  Solution  2  -f  5  drops  of  0.2  per  cent,  hydrochloric  acid. 

5.  Solution  2-1-5  drops  of  0.5  per  cent,  sodium  carbonate. 

6.  Solution  2  -f-  1  drop  of  36  per  cent,  acetic  acid. 

7.  Solution  2  -}-  1  drop  of  10  per  cent,  potassium  hydroxid. 

C.  Influence  of  associated  saline  matter. 

8.  Solution  2  -f  5  c.c.  of  5  per  cent,  sodium  chlorid. 

9.  Solution  2  -(-  5  c.c.  of  10  per  cent,  sodium  chlorid. 

328.  Subject  the  solutions  (1-9)  to  the  following  treatment  : 
Support  any  one  of  the  tubes  with  a  clamp  attached  to  the  iron  rod. 
Place  a  thermometer  in  the  clamped  tube  and  fasten,  witn  ::  rubber 
band,  all  the  other  tubes  to  the  one  in  the  clamp.  Immerse  the  tubes 
in  cold  w^aterin  a  large  beaker  on  a  tripod.  The  ends  of  the  tubes 
should  not  touch  the  bottom  of  the  beaker  and  the  level  of  the  liquid 
in  each  tube  should  be  below  the  level  of  the  surrounding  water.  Do 
not  transfer  the  thermometer  from  one  tube  to  another  during  the 
course  of  the  experiment.     It  may  be  safely  presumed  that  the  tem- 

*The  neutral  albumin  solutions  were  prepared  in  bulk  as  follows  :  Solutions 
of  egg  white  were  made  in  the  proportions  indicated  above  :  "  Dilute,"  1  in  40  ; 
"concentrated,"  1  in  10.  The  reaction  of  fresh  egg  white  is  slightly  though 
quite  distinctly  aZA;aMwe  (phosphates).  Each  of  the  two  solutions  was  carefully 
neutralized  with  very  dilute  acetic  acid,  warmed  to  the  temperature  of  the  body 
(37°  C.)  and  filtered. 


Proteins.  101 

perature  will  rise  uniformly  in  all  the  tubes.  Heat  the  water  in  the 
beaker  with  a  low  flame.  Stir  the  water  frequently.  Carry  the  tem- 
perature gradually  to  the  boiling  point  of  the  water  and  notice  the 
temperature  at  which  initial  coagulation  occurs,  i.  e.,  the  temperature 
at  which  the  faintest  possible  turbidity  can  be  detected.  Be  espe- 
cially alert  after  the  temperature  has  reached  50°  C  Coagulation 
will  begin  almost  simultaneously  in  several  of  the  tubes.  Do  not 
detach  any  of  the  tubes  until  after  the  surrounding  water  has  reached 
the  boiling  point. 

Salts  and  traces  of  free  acid  in  the  solvents  favor  coagulation  of 
proteins  that  are  coagulable  by  heat.  They  lower  the  temperature 
of  coagulation.  Heat  coagulation  is  impossible  in  the  absence  of 
salts.  It  is  prevented  by  alkali,  even  in  very  minute  proportions, 
and  also  by  acid  when  the  latter  is  present  in  appreciable  quantity. 
The  preventive  influence  of  alkalinity  is  greater  than  that  of  acid- 
ity.    No  proportion  of  alkalinity  is  favorable  to  the  process. 

Acid  or  alkali  converts  albumins  and  globulins  into  proteinates 
like  acidalbumin.  The  proteinates  are  noncoagulable  when  they 
are  dissolved  in  acid  or  alkaline  liquids,  but  they  may  be  coagulated, 
as  we  have  seen  (323),  if  heated  while  they  are  suspended  in  neu- 
tral fluids. 

Coagulated  protein  is  practically  insoluble  in  water,  saline  solu- 
tion, dilute  acid  and  dilute  alkali  (329). 

Acid  or  alkali,  when  present  in  cold  protein  solutions,  tends  to 
convert  coagulable  proteins  into  proteinates.  Heat  greatly  facili- 
tates the  process.  When  such  acid  or  alkaline  solutions  are  heated 
coagulable  proteins  may  be  entirely  converted  into  proteinate  before 
the  solution  reaches  the  temperature  of  coagulation  of  the  original 
protein.  When  the  proportion  of  acid  or  alkali  is  insufficient  to 
convert  all  of  the  coagulable  protein  into  proteinate  by  the  time 
the  coagulation  temperature  of  the  former  is  reached,  the  untrans- 
formed  portion  of  the  original  protein  will,  at  that  point  or  at  a 
somewhat  higher  temperature,  be  thrown  from  solution  as  floccu- 
lent  coagulum. 

If  coagulable  protein  is  dissolved  in  a  neutral  or  very  faintly  acid 
solution  (containing  saline  matter)  the  addition  of  a  slight  amount 
of  acid,  after  the  boiling  point  has  been  reached,  transforms  the  tur- 
bid opalescent  solution  to  a  flocculent  mixture  (324),  the  precipitated 


102  Biochemical,  Notes. 

protein  is  coagulated  in  the  process  and  an  excess  of  dilute  acid  or 
even  of  dilute  alkali  is  then  unable  to  dissolv'e  it. 

Demonstrations.  329.  Properties  of  coagulated  protein. 
330.  Method  for  the  quantitative  determination  of  coagulahle 
protein.  331.  Methods  for  the  detection  of  protein  in  the 
presence  of  fatty  products  and  carhohydrates.  332.  Methods 
for  the  separation  of  fats  and  fatty  products,  carbohydrates 
and  proteins  when  present  together  in  a  mixture. 

333.  Percentage  amounts  of  proteins  obtained  from  some 
mammalian  parts  and  from  various  vegetable  food-stuffs  (236).* 

The  figures  in  the  subjoined  table  represent  general  average  per- 
centage amounts  of  proteins  contained  in  the  materials  named  : 
Animal  parts.  Blood  : 

Urine Trace       Corpuscles 31.0 

Saliva 0.3       Plasma „.    8.0 

Gastric  juice 0.3   Tendon 34.7 

Adipose  tissue 0.9   Ligament 40.0 

Bile 2.5 

jlilk 4.0   Vegetable  foods. 

Lymph 4.1  Cucumber 1.0 

Liver 5.8  Potato 2.0 

Brain 7.3  Spinach 3.1 

Pancreatic  juice 8.0  Apple 4.0 

Bone  (shaft) 20.0  Eice  (grains) 7.0 

Cartilage 25.0  Wheat  (grains) 12.3 

Muscle 26.5  Peas  22.0 

*The  proportions  of  proteins  in  some  of  the  products  named  in  the  table  vary 
considerably  under  ordinary  conditions. 


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